Modified Insulin Polypeptides and Their Uses

ABSTRACT

Modified insulin polypeptides and their uses thereof are provided

FIELD OF THE INVENTION

This invention relates to insulin polypeptides optionally modified withat least one non-naturally encoded amino acid.

BACKGROUND OF THE INVENTION

Diabetes currently affects 246 million people worldwide and is expectedto affect 380 million by 2025 (International Diabetes Federationstatistic as of Nov. 15, 2007). Type 2 diabetes constitutes about 85% to95% of all diabetes cases in developed countries and accounts for aneven higher percentage in developing countries. The epidemic nature ofdiabetes continues to affect ever-increasing numbers of people aroundthe world while public awareness remains low.

Diabetes is any disorder characterized by excessive urine excretion. Themost common form of diabetes is diabetes mellitus, a metabolic disorderin which there is an inability to oxidize carbohydrate due todisturbances in insulin function. Diabetes mellitus is characterized byelevated glucose in the plasma and episodic ketoacidosis. Additionalsymptoms of diabetes mellitus include excessive thirst, glucosuria,polyuria, lipemia and hunger. If left untreated the disease can lead tofatal ketoacidosis. Other forms of diabetes include diabetes insipidusand brittle diabetes. Diabetes insipidus is the result of a deficiencyof antidiuretic hormone. The major symptom of diabetes insipidus(excessive urine output) results from an inability of the kidneys toresorb water. Brittle diabetes is a form that is very difficult tocontrol. It is characterized by unexplained oscillations betweenhypoglycemia and acidosis.

Criteria, which clinically establish an individual as suffering fromdiabetes mellitus, include: (1) having a fasting plasma glucose level inexcess of 126 mg/dL (7 mmol/L). Normal levels should be less than 100mg/dL (5.6 mmol/L) or (2) having plasma glucose levels in excess of 200mg/dL (11 mmol/L) at two times points during an oral glucose tolerancetest, OGTT, one of which must be within 2 hrs of ingestion of glucose.

The earlier a person is diagnosed with diabetes the better chance theperson has of staving off the primary negative consequences which arerenal failure, blindness and limb amputations due to circulatoryproblems. The American Diabetes Association has discussed recommendingthat physicians consider patients to be pre-diabetic if their fastingblood glucose level is above 100 mg/dL but less than 125 mg/dL and whoseglucose levels are at least 140 mg/dL but less than 200 mg/dL followingan oral glucose tolerance test (OGTT).

Diabetes mellitus is a heterogeneous clinical disorder with numerouscauses. Two main classifications of diabetes mellitus exist, idiopathicand secondary. Idiopathic diabetes is divided into two main types;insulin dependent and non-insulin-dependent. Insulin-dependent diabetesmellitus, IDDM (more commonly referred to as type 1 diabetes) is definedby the development of ketoacidosis in the absence of insulin therapy.Type 1 diabetes most often manifests in childhood (hence also calledjuvenile onset diabetes) and is the result of an autoimmune destructionof the b-cells of the pancreas. Non-insulin-dependent diabetes mellitus,NIDDM (more commonly referred to as type 2 diabetes) is characterized bypersistent hyperglycemia but rarely leads to ketoacidosis. Type 2diabetes generally manifests after age 40 and therefore has the obsoletename of adult onset-type diabetes. Type 2 diabetes can result fromgenetics defects that cause both insulin resistance and insulindeficiency. There are two main forms of type 2 diabetes: (1) late onsetassociated with obesity; and (2) late onset not associated with obesity.Many, if not all, of the negative long term effects of living with type2 diabetes are due to persistent hyperglycemia. For this reason a majorgoal of therapeutic intervention in type 2 diabetes is to reducecirculating glucose levels.

The major function of insulin is to counter the concerted action of anumber of hyperglycemia-generating hormones and to maintain low bloodglucose levels. Because there are numerous hyperglycemic hormones,untreated disorders associated with insulin generally lead to severehyperglycemia and shortened life span.

In addition to its role in regulating glucose metabolism, insulinstimulates lipogenesis, diminishes lipolysis, and increases amino acidtransport into cells. Insulin also modulates transcription, altering thecell content of numerous mRNAs. It stimulates growth, DNA synthesis, andcell replication, effects that it holds in common with the insulin-likegrowth factors (IGFs) and relaxin.

SUMMARY OF THE INVENTION

The invention provides insulin polypeptides optionally modified with atleast one non-naturally encoded amino acid. This specification willprovide some embodiments, however it should be appreciated that theseembodiments are for the purpose of illustrating the invention, and arenot to be construed as limiting the scope of the invention as defined bythe claims.

In non-diabetic individuals, the pancreas continuously supplies low (orbasal) levels of insulin. The liver supplies glucose to the rest of thebody when the body is in a fasting state. This is typically referred toas hepatic glucose production. In response to a meal, high levels ofinsulin are released by the pancreas. The insulin first interacts withthe liver, signaling the liver to stop hepatic glucose production and tobegin absorbing glucose ingested as part of the meal. Some of the bolusof insulin release by the pancreas passes through the liver andinteracts with other body cells, especially muscle, signaling them toabsorb and use glucose. In a diabetic, however, this tandem work done bythe pancreas and the liver may be disrupted and it is desirable toprovide a therapy for diabetic patients that would help them maintainglucose within healthy limits without experiencing hyper or hypoglycemia and the associated short and long term effects of thesefluctuations. Therefore, it has long been a goal of insulin therapy tomimic the pattern of endogenous insulin secretion in normal individuals.The daily physiological demand for insulin fluctuates and can beseparated into two phases: (a) the absorptive phase requiring a pulse ofinsulin to dispose of the meal-related blood glucose surge, and (b) thepost-absorptive phase requiring a sustained amount of insulin toregulate hepatic glucose output for maintaining optimal fasting bloodglucose. Accordingly, effective therapy generally involves the combineduse of two exogenous insulins: a fast-acting meal time insulin providedby bolus injections and a long-acting basal insulin administered byinjection once or twice daily.

In a patent application assigned to Eli Lilly, U.S. Publication No.20040242460, incorporated herein by reference, a class of acylatedinsulins was disclosed for use as a long-acting basal insulin therapy.The acylated insulins are prepared by acylating, selectively with anactivated fatty acid derivative, the free amino group(s) of a monomericinsulin, including normal insulin and certain insulin analogs. Usefulfatty acid derivatives include reactive fatty acid-type compounds havingat least a six (6) carbon atom chain length and particularly those fattyacid derivatives having 8 to 21 carbon atoms in their chain.Mono-acylated normal human insulin, acylated with a palmitic acidderivative, is a particularly promising candidate. Insulins fallingwithin this category are described in Japanese patent application1-254,699. An embodiment of the present invention provides for acylatedinsulins containing one or more non-natural amino acids. An additionalembodiment of the present invention includes acylated insulins with oneor more non-naturally encoded amino acids linked to a water solublepolyer, such as PEG.

Methods according to embodiments of the present invention may restoreglucose homeostasis to a diabetic individual having a healthy liver(i.e., a liver capable of normal glucose uptake and production but forthe absence of the regulating hormone insulin). By activating the liverto regulate the level of blood glucose, methods of the present inventionmay reduce or eliminate the hyperglycemia and/or hypoglycemia associatedwith conventional methods of treatment of diabetes mellitus. Methodsaccording to embodiments of the present invention may also reduce oreliminate some, if not all, of the microvascular complications (e.g.,nephropathy, retinopathy and/or neuropathy) and/or macrovascularcomplications (e.g., myocardial infarction and/or stroke) typicallyassociated with diabetes mellitus. Moreover, methods according toembodiments of the present invention may reduce or eliminate thehyperinsulinemia associated with the peripheral administration (e.g.,subcutaneous, intrapulmonary, intranasal, buccal mucosal) of insulin.Furthermore, methods according to embodiments of the present inventionmay reduce or eliminate the hyperlipidemia associated with diabetes byactivating the liver to improve its fatty acid metabolism. Properactivation of the liver may also restore other liver cell,gene-regulated metabolic pathways related to complications associatedwith diabetes mellitus.

In some embodiments, the insulin polypeptide comprises one or morepost-translational modifications. In some embodiments, the insulinpolypeptide is linked to a linker, polymer, or biologically activemolecule. In some embodiments, the insulin polypeptide is linked to abifunctional polymer, bifunctional linker, or at least one additionalinsulin polypeptide.

In some embodiments, the non-naturally encoded amino acid is linked to awater soluble polymer. In some embodiments, the water soluble polymercomprises a poly(ethylene glycol) moiety. In some embodiments, thenon-naturally encoded amino acid is linked to the water soluble polymerwith a linker or is bonded to the water soluble polymer. In someembodiments, the poly(ethylene glycol) molecule is a bifunctionalpolymer. In some embodiments, the bifunctional polymer is linked to asecond polypeptide. In some embodiments, the second polypeptide is aninsulin polypeptide.

In some embodiments, the insulin polypeptide comprises at least twoamino acids linked to a water soluble polymer comprising a poly(ethyleneglycol) moiety. In some embodiments, at least one amino acid is anon-naturally encoded amino acid.

In some embodiments, the insulin polypeptide comprises at least twoamino acids linked to a water soluble polymer comprising a poly(ethyleneglycol) moiety. In some embodiments, at least one amino acid is anon-naturally encoded amino acid.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: in the A chain beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., at the carboxylterminus of the protein), and any combination thereof (SEQ ID NO: 1 orthe corresponding amino acids in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, or SEQ ID NO: 11) and/or in the 13 chain beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31 (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NO: 4,SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulin:before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 (i.e., at thecarboxyl terminus of the protein of SEQ ID NO: 13 or the correspondingamino acids in SEQ ID NO: 14). In some embodiments, one or morenon-naturally encoded amino acids are incorporated in one or more of thefollowing positions in any of the insulin, insulin analogs, insulinlispro, insulin glulisine, insulin detemir, or insulin glarginepolypeptides: in the A chain 1, 9, 14, 15 (SEQ ID NO: 1 or thecorresponding amino acid positions in SEQ ID NOs: 3, 5, 7, 9, 11). Insome embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: in the B chain 1, 22, 28 (SEQID NO: 2 or the corresponding amino acid positions in SEQ ID NOs: 2, 4,6, 8, 10, 12). In some embodiments, one or more non-naturally encodedamino acids are incorporated in one or more of the following positionsin any of the insulin, insulin analogs, insulin lispro, insulinglulisine, insulin detemir, or insulin glargine polypeptides: in the Achain 1, 4, 5, 8, 9, 12, 14, 15, 18 (SEQ ID NO: 1 or the correspondingamino acid positions in SEQ ID NOs: 3, 5, 7, 9, 11). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: in the B chain 1, 3, 4, 21,22, 28, 29 (SEQ ID NO: 2 or the corresponding amino acid positions inSEQ ID NOs: 2, 4, 6, 8, 10, 12).

In some embodiments, one or more non-naturally encoded amino acids areincorporated at one or more of the following positions of insulin or aninsulin analog: A chain positions 8, 9, 10, 14 (SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11); chainpositions 1, 17, 21, 25, 28 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12). In some embodiments, one ormore non-naturally encoded amino acids are incorporated at one or moreof the following positions of insulin: A chain positions 8, 9, 10, 14(SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11); chain positions 1, 17, 21, 25, 28 (SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12). Insome embodiments, one or more non-naturally encoded amino acids areincorporated at one or more of the following positions of insulinanalog: A chain positions 8, 9, 10, 14 (SEQ ID NO: 1, SEQ ID NO: 3, SEQID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11); chain positions 1,17, 21, 25, 28 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12).

In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is linked to a water soluble polymer, including butnot limited to, positions: in the A chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of the protein), andany combination thereof (SEQ ID NO: 1 or the corresponding amino acidsin SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO:11) and/or in the B chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 2 or thecorresponding amino acids in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12). In some embodiments, the non-naturallyencoded amino acid at one or more of these positions is linked to awater soluble polymer, including but not limited to: before position 1(i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111 (i.e., at the carboxyl terminusof the protein of SEQ ID NO: 13 or the corresponding amino acids in SEQID NO: 14). In some embodiments, the non-naturally encoded amino acid atone or more of these positions is linked to a water soluble polymer,including but not limited to: in the A chain 1, 4, 5, 8, 9, 12, 14, 15,18 (SEQ ID NO: 1 or the corresponding amino acid positions in SEQ IDNOs: 3, 5, 7, 9, 11). In some embodiments, the non-naturally encodedamino acid at one or more of these positions is linked to a watersoluble polymer, including but not limited to: in the B chain 1, 3, 4,21, 22, 28, 29 (SEQ ID NO: 2 or the corresponding amino acid positionsin SEQ ID NOs: 2, 4, 6, 8, 10, 12).

In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is linked to a water soluble polymer, including butnot limited to, positions: A chain positions 8, 9, 10, 14 (SEQ ID NO: 1,SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11);chain positions 1, 17, 21, 25, 28 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12). In some embodiments,the non-naturally encoded amino acid at one or more of these positionsis linked to a water soluble polymer, including but not limited to,positions: A chain positions 8, 9, 10, 14 (SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11); chainpositions 1, 17, 21, 25, 28 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12). In some embodiments, thenon-naturally encoded amino acid at one or more of these positions islinked to a water soluble polymer, including but not limited to,positions: A chain positions 8, 9, 10, 14 (SEQ ID NO: 1, SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11); B chainpositions 1, 17, 21, 25, 28 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12).

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: B chain positions 1, 2, 9,10, 28, 29, 30 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12). In some embodiments, the non-naturallyencoded amino acid at one or more of these positions is linked to awater soluble polymer, including but not limited to, positions: B chainpositions 1, 2, 9, 10, 28, 29, 30 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12).

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: A chain 21, or B chainpositions 1, 10, 13, 16, 28, 29, 31, or 32 (i.e., at the carboxylterminus of the protein) (SEQ ID NO:1 through SEQ ID NO: 12). In someembodiments, the non-naturally encoded amino acid at one or more ofthese positions is linked to a water soluble polymer, including but notlimited to, positions: A chain 21, or B chain positions 1, 10, 13, 16,28, 29, 31, or 32 (i.e., at the carboxyl terminus of the protein) (SEQID NO:1 through SEQ ID NO: 12).

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: A chain positions 8, 9, 10,14 (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO:9, SEQ ID NO: 11). In some embodiments, the non-naturally encoded aminoacid at one or more of these positions is linked to a water solublepolymer, including but not limited to, positions: A chain positions 8,9, 10, 14 (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11).

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: B chain positions 17, 21, 25,28 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12). In some embodiments, the non-naturally encoded aminoacid at one or more of these positions is linked to a water solublepolymer, including but not limited to, positions: B chain positions 17,21, 25, 28 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12).

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: A chain positions 4, 8, 9,10, 14, 18, 21 (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11). In some embodiments, the non-naturallyencoded amino acid at one or more of these positions is linked to awater soluble polymer, including but not limited to, positions: A chainpositions 4, 8, 9, 10, 14, 18, 21 (SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11).

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in any of theinsulin, insulin analogs, insulin lispro, insulin glulisine, insulindetemir, or insulin glargine polypeptides: B chain positions 1, 5, 17,21, 25, 29, 30 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12). In some embodiments, the non-naturallyencoded amino acid at one or more of these positions is linked to awater soluble polymer, including but not limited to, positions: B chainpositions 1, 5, 17, 21, 25, 29, 30 (SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12).

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulin: inthe A chain before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., atthe carboxyl terminus of the protein), and any combination thereof (SEQID NO: 1) or in the B chain before position 1 (i.e. at the N-terminus),1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 2). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulinlispro: in the A chain before position 1 (i.e. at the N-terminus), 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22(i.e., at the carboxyl terminus of the protein), and any combinationthereof (SEQ ID NO: 3) or in the B chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 4).In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulinaspart: in the A chain before position 1 (i.e. at the N-terminus), 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22(i.e., at the carboxyl terminus of the protein), and any combinationthereof (SEQ ID NO: 5) or in the B chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 6).In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulinglulisine: in the A chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22 (i.e., at the carboxyl terminus of the protein), and any combinationthereof (SEQ ID NO: 7) or in the B chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 8).In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulindetemir: in the A chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22 (i.e., at the carboxyl terminus of the protein), and any combinationthereof (SEQ ID NO: 9) or in the B chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 10).In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulinglargine: in the A chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22 (i.e., at the carboxyl terminus of the protein), and any combinationthereof (SEQ ID NO: 11) or in the B chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 12).

In some embodiments, the non-naturally encoded amino acid at one or moreof these positions in any of the insulin, insulin lispro, insulinglulisine, insulin detemir, or insulin glargine polypeptides is linkedto a water soluble polymer, including but not limited to, positions:before position 1 (i.e. at the N-terminus), in the A chain beforeposition 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., at the carboxylterminus of the protein), and any combination thereof (SEQ ID NO: 1 orthe corresponding amino acids in SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, or SEQ ID NO: 11) or in the B chain before position 1(i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQID NO: 2 or the corresponding amino acids in SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12).

In some embodiments, the non-naturally encoded amino acid at one or moreof these positions in the insulin polypeptide is linked to a watersoluble polymer, including but not limited to, positions: beforeposition 1 (i.e. at the N-terminus), in the A chain before position 1(i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of theprotein), and any combination thereof (SEQ ID NO: 1) or in the B chainbefore position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31 (SEQ ID NO: 2). In some embodiments, the non-naturallyencoded amino acid at one or more of these positions in the insulinlispro polypeptide is linked to a water soluble polymer, including butnot limited to, positions: in the A chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of the protein), andany combination thereof (SEQ ID NO: 3) or in the B chain before position1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31(SEQ ID NO: 4). In some embodiments, the non-naturally encoded aminoacid at one or more of these positions in the insulin aspart polypeptideis linked to a water soluble polymer, including but not limited to,positions: in the A chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22 (i.e., at the carboxyl terminus of the protein), and any combinationthereof (SEQ ID NO: 5) or in the B chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 6).In some embodiments, the non-naturally encoded amino acid at one or moreof these positions in the insulin glulisine polypeptide is linked to awater soluble polymer, including but not limited to, positions: in the Achain before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., at thecarboxyl terminus of the protein), and any combination thereof (SEQ IDNO: 7) or in the B chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 8). In someembodiments, the non-naturally encoded amino acid at one or more ofthese positions in the insulin detemir polypeptide is linked to a watersoluble polymer, including but not limited to, positions: in the A chainbefore position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., at thecarboxyl terminus of the protein), and any combination thereof (SEQ IDNO: 9) or in the B chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 10). In someembodiments, the non-naturally encoded amino acid at one or more ofthese positions in the insulin glargine polypeptide is linked to a watersoluble polymer, including but not limited to, positions: in the A chainbefore position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., at thecarboxyl terminus of the protein), and any combination thereof (SEQ IDNO: 11) or in the B chain before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 12).

In some embodiments, one or more non-naturally encoded amino acids areincorporated at one or more of the following positions of any of theinsulin, insulin lispro, insulin glulisine, insulin detemir, or insulinglargine polypeptides: 28, 36, 76, 80, 107, 108, 111, 8, 15, 19, 36, 42,46, 48, 49, 80, 108, 111, 113, 155, and any combination thereof (SEQ IDNO: 1 and SEQ ID NO: 2; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID NO: 5 andSEQ ID NO: 6; SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 9 and SEQ ID NO:10; or SEQ ID NO: 11 and SEQ ID NO: 12).

Methods of the present invention could be used to produce crystals ofinsulin analogs containing one or more non-naturally encoded aminoacids. U.S. Pat. No. 7,193,035 assigned to Sanofi-Aventis DeutschlandGmbH, which is hereby incorporated by reference, discloses formation anduse of crystals of an insulin analog.

Formulations

In the broad practice of the present invention, it also is contemplatedthat a formulation may contain a mixture of two or more of an insulin,an insulin analog, an acylated insulin, or acylated insulin analog withat least one of the components of the mixture containing a non-naturallyencoded amino acid. In another embodiment of the present invention, theformulations containing a mixture of two or more of insulin, an insulinanalog, an acylated insulin, or acylated insulin analog with at leastone of the components of the mixture containing a non-naturally encodedamino acid also includes at least one water soluble polymer attached toat least one of the non-naturally encoded amino acids.

The present invention also includes heterogenous mixtures whereininsulin polypeptides and insulin analogs are prepared by the methodsdisclosed in this invention and are then mixed so that a formulation maybe administered to a patient in need thereof which contains, forexample, 25% insulin polypeptide containing a non-naturally encodedamino acid at position 28 of the B chain which has been pegylated, 25%insulin polypeptide containing a non-naturally encoded amino acid atposition 10 of the B chain, said non-naturally encoded amino acidcoupled to a water soluble polymer, and 50% insulin polypeptide whereina non-naturally encoded amino acid occurs at position 31 of the B chainof insulin (SEQ ID NO: 2; alternatively SEQ ID NOs: 4, 6, 8, 10, or 12).All different mixtures of different percentage amounts of insulinpolypeptide variants wherein the insulin polypeptides include a variety(1) with differently sized PEGs, or (2) PEGS are included at differentpositions in the sequence. This is intended as an example and should inno way be construed as limiting to the formulations made possible by thepresent invention and will be apparent to those of skill in the art. Inan additional embodiment, the insulin polypeptide variants to include inthe formulation mixture will be chosen by their varying dissociationtimes so that the formulation may provide a sustained release of insulinfor a patient in need thereof.

Formulations of the present invention may include a glucagon.

Other Embodiments of the Present Invention Including Formulation forInhalation

In an additional embodiment of the present invention, it is possible touse the technology disclosed herein for the production of insulinanalogs with increased pharmacokinetic and pharacodynamic properties forpatient use via administration to the lung, resulting in elevated bloodlevels of insulin that are sustained for at least 6 hours, and moretypically for at least 8, 10, 12, 14, 18, 24 hours or greaterpost-administration. Another embodiment of the present invention allowsfor advantageous mixtures of insulin analogs suitable for therapeuticformulations designed to be administered to patients as an inhalant.

In some embodiments of the present invention, the following sites in thenative insulin molecule may be substituted with non-naturally encodedamino acids and optionally further modified by covalent attachment of awater soluble polymer, such as PEG: the 2 C-termini of the A and Bchains, Arg22B, His10B, His5A, Glu4A, Glu17A, Glu13B, and Glu21B.

In addition to native insulin, the present invention provides fornon-native insulin polypeptides and insulin analogs having one or morenon-naturally encoded amino acids substituted or inserted into thesignal sequence that may also provide a site for the incorporation ofone or more water soluble polymers, such as PEG. This embodiment of theinvention is particularly useful for introducing additional, customizedpegylation-sites within the insulin molecule, for example, for forming aPEG-insulin having improved resistance to enzymatic degradation. Such anapproach provides greater flexibility in the design of an optimizedinsulin conjugate having the desired balance of activity, stability,solubility, and pharmacological properties. Mutations can be carriedout, i.e., by site specific mutagenesis, at any number of positionswithin the insulin molecule. PEGs for use in the present invention maypossess a variety of structures: linear, forked, branched, dumbbell, andthe like. Typically, PEG is activated with a suitable activating groupappropriate for coupling a desired site or sites on the insulinmolecule. An activated PEG will possess a reactive group at a terminusfor reaction with insulin. Representative activated PEG derivatives andmethods for conjugating these agents to a drug such as insulin are knownin the art and further described in Zalipsky, S., et al., “Use ofFunctionalized Poly(Ethylene Glycols) for Modification of Polypeptides”in Polyethylene Glycol Chemistry: Biotechnical and BiomedicalApplications, J. M. Harris, Plenus Press, New York (1992), and inAdvanced Drug Reviews, 16:157-182 (1995).

In one particular embodiment of the invention, the PEG portion of theconjugate is absent one or more lipophilic groups effective tosignificantly modify the water-soluble nature of the polymer or of thepolymer-insulin conjugate. That is to say, the polymer or non-insulinportion of a conjugate of the invention may contain a group of atomsconsidered to be more lipophilic than hydrophilic (e.g., a carbon chainhaving from about 2 to 8-12 carbon atoms), however, if the presence ofsuch a group or groups is not effective to significantly alter thehydrophilic nature of the polymer or of the conjugate, then such amoiety may be contained in the conjugates of the invention. That is tosay, through site-specific mutations of insulin, insulin polypeptides,and insulin analogs, an insulin conjugate of the invention itself mayexhibit hydrophilic, rather than lipophilic or amphiphilic. In certainembodiments of the invention where a lipophilic moiety may be present,the moiety is preferably not positioned at a terminus of a PEG chain.

Branched PEGs for use in the conjugates of the invention include thosedescribed in International Patent Publication WO 96/21469, the contentsof which is expressly incorporated herein by reference in its entirety.Generally, branched PEGs can be represented by the formulaR(PEG-OH).sub.n, where R represents the central “core” molecule and.sub.n represents the number of arms. Branched PEGs have a central corefrom which extend 2 or more “PEG” arms. In a branched configuration, thebranched polymer core possesses a single reactive site for attachment toinsulin. Branched PEGs for use in the present invention will typicallycomprise fewer than 4 PEG arms, and more preferably, will comprise fewerthan 3 PEG arms. Branched PEGs offer the advantage of having a singlereactive site, coupled with a larger, more dense polymer cloud thantheir linear PEG counterparts. One particular type of branched PEG canbe represented as (MeO-PEG-).sub.p R—X, where p equals 2 or 3, R is acentral core structure such as lysine or glycerol having 2 or 3 PEG armsattached thereto, and X represents any suitable functional group that isor that can be activated for coupling to insulin. One particularlypreferred branched PEG is mPEG2-NHS (Shearwater Corporation, Alabama)having the structure mPEG2-lysine-succinimide.

In yet another branched architecture, “pendant PEG” has reactive groupsfor protein coupling positioned along the PEG backbone rather than atthe end of PEG chains. The reactive groups extending from the PEGbackbone for coupling to insulin may be the same or different. PendantPEG structures may be useful but are generally less preferred,particularly for compositions for inhalation.

Alternatively, the PEG-portion of a PEG-insulin conjugate may possess aforked structure having a branched moiety at one end of the polymerchain and two free reactive groups (or any multiple of 2) linked to thebranched moiety for attachment to insulin. Exemplary forked PEGS aredescribed in International Patent Publication No. WO 99/45964, thecontent of which is expressly incorporated herein by reference. Theforked polyethylene glycol may optionally include an alkyl or “R” groupat the opposing end of the polymer chain. More specifically, a forkedPEG-insulin conjugate in accordance with the invention has the formula:R-PEG-L(Y-insulin)n where R is alkyl, L is a hydrolytically stablebranch point and Y is a linking group that provides chemical linkage ofthe forked polymer to insulin, and n is a multiple of 2. L may representa single “core” group, such as “—CH—”, or may comprise a longer chain ofatoms. Exemplary L groups include lysine, glycerol, pentaerythritol, orsorbitol. Typically, the particular branch atom within the branchingmoiety is carbon.

In one particular embodiment of the invention, the linkage of the forkedPEG to the insulin molecule, (Y), is hydrolytically stable. In apreferred embodiment, n is 2. Suitable Y moieties, prior to conjugationwith a reactive site on insulin, include but are not limited to activeesters, active carbonates, aldehydes, isocyanates, isothiocyanates,epoxides, alcohols, maleimides, vinylsulfones, hydrazides,dithiopyridines, and iodacetamides. Selection of a suitable activatinggroup will depend upon the intended site of attachment on the insulinmolecule and can be readily determined by one of skill in the art. Thecorresponding Y group in the resulting PEG-insulin conjugate is thatwhich results from reaction of the activated forked polymer with asuitable reactive site on insulin. The specific identity of such thefinal linkage will be apparent to one skilled in the art. For example,if the reactive forked PEG contains an activated ester, such as asuccinimide or maleimide ester, conjugation via an amine site on insulinwill result in formation of the corresponding amide linkage. Theseparticular forked polymers are particularly attractive since theyprovide conjugates having a molar ratio of insulin to PEG of 2:1 orgreater. Such conjugates may be less likely to block the insulinreceptor site, while still providing the flexibility in design toprotect the insulin against enzymatic degradation, e.g., by insulindegrading enzyme.

In a related embodiment, a forked PEG-insulin conjugate may be used inthe present invention, represented by the formula:R-[PEG-L(Y-insulin)2]n. In this instance R represents a non-naturallyencoded amino acid having attached thereto at least one PEG-di-insulinconjugate. Specifically, preferred forked polymers in accordance withthis aspect of the invention are those were n is selected from the groupconsisting of 1, 2, 3, 4, 5, and 6. In an alternative embodiment, thechemical linkage between the non-natural amino acid within insulin,insulin polypeptide, or insulin analog and the polymer branch point maybe degradable (i.e., hydrolytically unstable). Alternatively, one ormore degradable linkages may be contained in the polymer backbone toallow generation in vivo of a PEG-insulin conjugate having a smaller PEGchain than in the initially administered conjugate. For example, a largeand relatively inert conjugate (i.e., having one or more high molecularweight PEG chains attached thereto, e.g., one or more PEG chains havinga molecular weight greater than about 10,000, wherein the conjugatepossesses essentially no bioactivity) may be administered, which theneither in the lung or in the bloodstream, is hydrolyzed to generate abioactive conjugate possessing a portion of the originally present PEGchain. Upon in-vivo cleavage of the hydrolytically degradable linkage,either free insulin (depending upon the position of the degradablelinkage) or insulin having a small polyethylene tag attached thereto, isthen released and more readily absorbed through the lung and/orcirculated in the blood.

In one feature of this embodiment of the invention, the intactpolymer-conjugate, prior to hydrolysis, is minimally degraded uponadministration, such that hydrolysis of the cleavable bond is effectiveto govern the slow rate of release of active insulin into thebloodstream, as opposed to enzymatic degradation of insulin prior to itsrelease into the systemic circulation.

Appropriate physiologically cleavable linkages include but are notlimited to ester, carbonate ester, carbamate, sulfate, phosphate,acyloxyalkyl ether, acetal, and ketal. Such conjugates should possess aphysiologically cleavable bond that is stable upon storage and uponadministration. For instance, a PEG-cleavable linkage-insulin conjugateshould maintain its integrity upon manufacturing of the finalpharmaceutical composition, upon dissolution in an appropriate deliveryvehicle, if employed, and upon administration irrespective of route.

Single-Chain Insulin

When focussing on peripheral insulin resistance, the drug of choice is athiazolidinedione, which is a type of insulin-sensitizing agent.Troglitazone (TRG), for example, is an orally active anti-diabetic agentof the thiazolidinedione chemical series. This drug has been shown toreverse insulin resistance in patients with NIDDM and impaired glucosetolerance, and can enhance insulin action in numerous genetic andacquired rodent models of insulin resistance. The antihyperglycemiceffects of TRG result from its ability to increase insulin dependentglucose disposal and reduce hepatic glucose production. It is believed,by enhancing insulin action, TRG treatment results in euglycemia at alower circulating insulin level. In this regard, studies in normal anddiabetic rodents and human clinical trials have not revealedhypoglycemia as a complication of thiazolidinedione therapy. On theother hand, administration of these drugs to normal or insulin-deficientdiabetic animals failed to alter plasma glucose or insulin or glucosetolerance, although insulin sensitivity was nevertheless increased.

The two chain structure of insulin allows insulin to undertake multipleconformations, and several findings have indicated that insulin has thepropensity to considerable conformational change and that restrictionsin the potential for such change considerably decrease the affinity ofthe insulin receptor for ligands. Proinsulin has a 100 fold loweraffinity for the insulin receptor than native insulin. Blocking of theamino acid residue A1 in insulin also results in poor receptor binding,consistent with the dogma that a free N-terminal of the A-chain and freeC-terminal of the B-chain of insulin are important for binding to theinsulin receptor.

The inherited physical and chemical stability of the insulin molecule isa basic condition for insulin therapy of diabetes mellitus. These basicproperties are fundamental for insulin formulation and for applicableinsulin administration methods, as well as for shelf-life and storageconditions of pharmaceutical preparations. Use of solutions inadministration of insulin exposes the molecule to a combination offactors, e.g. elevated temperature, variable air-liquid-solidinterphases as well as shear forces, which may result in irreversibleconformation changes e.g. fibrillation. This is particularly relevantfor insulin solutions in infusion pumps, either worn externally orimplanted, which exposes the molecule to a combination of these factorsas well as shear forces from the movement of the pump for extendedperiods of time. Consequently, fibrillation is especially a concern whenusing infusion pumps as insulin delivery system. Moreover, thesolubility of insulin is influenced by multiple factors and shows clearreduction in the pH range from 4.2 and 6.6. The pH precipitation zonegenerally imposes limitations for formulation, but has also been useddeliberately in development and formulation of certain analogues.

Thus, in another embodiment of the present invention, one or morenon-naturally encoded amino acids are incorporated into a single chaininsulin or single chain insulin analog. Single-chain insulins withinsulin activity are disclosed in EP 1,193,272. These single-chaininsulins have a modified C-peptide of 5-18 amino acids and are reportedto have up to 42% insulin activity. EP 1,193,272 discloses the followingmodified C-peptides connecting B30 with A21:Gly-Gly-Gly-Pro-Gly-Lys-Arg, Arg-Arg-Gly-Pro-Gly-Gly-Gly,Gly-Gly-Gly-Gly-Gly-Lys-Arg, Arg-Arg-Gly-Gly-Gly-Gly-Gly,Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg,Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly,Gly-Gly-Tyr-Pro-Gly-Asp-Val-Lys-Arg,Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly,Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg, andArg-Arg-His-Pro-Gly-Asp-Val-Gly-Gly. EP 741,188 discloses single-chaininsulins with a modified C-peptide having from 10-14 amino acidsresidues and having from 14 to 34% insulin activity and having thefollowing connecting peptidesGln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg andGly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr. WO 95/16708 disclosessingle-chain insulins with a connecting peptide of 1-15 amino acidresidues and with no Lys or Arg as the C-terminal amino acid residue inthe connecting peptide. WO 95/16708 discloses the following C-peptidesequences Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr andGly-Tyr-Gly-Ser-Ser-Ser-Ala-Ala-Ala-Pro-Gln-Thr. These single-chaininsulins are reported to have insulin activity but also a fairly highaffinity to the IGF-1 receptor.

Novo Nordisk, Inc. U.S. Patent Publication No. 20070129284 disclosessingle chain insulin analogs by introduction of a C-peptide between theB- and A-chain to decrease molecular flexibility and concomitantlyreduce the fibrillation propensity and limit or modify the pHprecipitation zone. One example of the single-chain insulin disclosed byNovo Nordisk, Inc., comprises the B- and the A-chain of human insulin oranalogues or derivatives thereof connected by a connecting peptide,wherein the connecting peptide has from 5-11 amino acid residuesprovided that if the connecting peptide contains two adjacent basicamino acid residues then at least one of the natural amino acid residuesin the B and/or A chain is substituted with another codable amino acidresidue or at least one lysine residue in the A-chain, in the B-chain orin the connecting peptide has been chemically modified by acylation orthe connecting peptide is not one of the following sequencesGly-Gly-Gly-Pro-Gly-Lys-Arg, Arg-Arg-Gly-Pro-Gly-Gly-Gly,Gly-Gly-Gly-Gly-Gly-Lys-Arg, Arg-Arg-Gly-Gly-Gly-Gly-Gly,Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg,Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly,Gly-Gly-Tyr-Pro-Gly-Asp-Val-Lys-Arg,Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly,Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg, orArg-Arg-His-Pro-Gly-Asp-Val-Gly-Gly.

In some embodiments, the polypeptide of the invention comprises one ormore non-naturally encoded amino acid substitution, addition, ordeletion in the signal sequence. In some embodiments, the polypeptide ofthe invention comprises one or more non-naturally encoded amino acidsubstitution, addition, or deletion in the signal sequence for insulinor any of the insulin analogs or polypeptides disclosed within thisspecification. In some embodiments, the polypeptide of the inventioncomprises one or more naturally encoded amino acid substitution,addition, or deletion in the signal sequence as well as one or morenon-naturally encoded amino acid substitutions, additions, or deletionsin the signal sequence for insulin or any of the insulin analogs orpolypeptides disclosed within this specification. In some embodiments,one or more non-natural amino acids are incorporated in the leader orsignal sequence for insulin or any of the insulin analogs orpolypeptides disclosed within this specification.

In some embodiments, the insulin polypeptide comprises a substitution,addition or deletion that modulates affinity of the insulin polypeptidefor an insulin polypeptide receptor or binding partner, including butnot limited to, a protein, polypeptide, small molecule, or nucleic acid.In some embodiments, the insulin polypeptide comprises a substitution,addition, or deletion that increases the stability of the insulinpolypeptide when compared with the stability of the correspondinginsulin without the substitution, addition, or deletion. Stabilityand/or solubility may be measured using a number of different assaysknown to those of ordinary skill in the art. Such assays include but arenot limited to SE-HPLC and RP-HPLC. In some embodiments, the insulinpolypeptide comprises a substitution, addition, or deletion thatmodulates the immunogenicity of the insulin polypeptide when comparedwith the immunogenicity of the corresponding insulin without thesubstitution, addition, or deletion. In some embodiments, the insulinpolypeptide comprises a substitution, addition, or deletion thatmodulates serum half-life or circulation time of the insulin polypeptidewhen compared with the serum half-life or circulation time of thecorresponding insulin without the substitution, addition, or deletion.

In some embodiments, the insulin polypeptide comprises a substitution,addition, or deletion that increases the aqueous solubility of theinsulin polypeptide when compared to aqueous solubility of thecorresponding insulin without the substitution, addition, or deletion.In some embodiments, the insulin polypeptide comprises a substitution,addition, or deletion that increases the solubility of the insulinpolypeptide produced in a host cell when compared to the solubility ofthe corresponding insulin without the substitution, addition, ordeletion. In some embodiments, the insulin polypeptide comprises asubstitution, addition, or deletion that increases the expression of theinsulin polypeptide in a host cell or increases synthesis in vitro whencompared to the expression or synthesis of the corresponding insulinwithout the substitution, addition, or deletion. The insulin polypeptidecomprising this substitution retains agonist activity and retains orimproves expression levels in a host cell. In some embodiments, theinsulin polypeptide comprises a substitution, addition, or deletion thatincreases protease resistance of the insulin polypeptide when comparedto the protease resistance of the corresponding insulin without thesubstitution, addition, or deletion. In some embodiments, the insulinpolypeptide comprises a substitution, addition, or deletion thatmodulates signal transduction activity of the insulin receptor whencompared with the activity of the receptor upon interaction with thecorresponding insulin polypeptide without the substitution, addition, ordeletion. In some embodiments, the insulin polypeptide comprises asubstitution, addition, or deletion that modulates its binding toanother molecule such as a receptor when compared to the binding of thecorresponding insulin polypeptide without the substitution, addition, ordeletion. In some embodiments, the insulin polypeptide comprises asubstitution, addition, or deletion that modulates its anti-viralactivity compared to the anti-viral activity of the correspondinginsulin polypeptide without the substitution, addition, or deletion. Insome embodiments, the insulin polypeptide comprises a substitution,addition, or deletion that enhances its glucose metabolizing activitycompared to the glucose metabolizing activity of the correspondinginsulin polypeptide without the substitution, addition, or deletion.

In some embodiments, the insulin polypeptide comprises a substitution,addition, or deletion that increases compatibility of the insulinpolypeptide with pharmaceutical preservatives (e.g., m-cresol, phenol,benzyl alcohol) when compared to compatibility of the correspondinginsulin without the substitution, addition, or deletion. This increasedcompatibility would enable the preparation of a preserved pharmaceuticalformulation that maintains the physiochemical properties and biologicalactivity of the protein during storage.

In some embodiments, one or more engineered bonds are created with oneor more non-natural amino acids. The intramolecular bond may be createdin many ways, including but not limited to, a reaction between two aminoacids in the protein under suitable conditions (one or both amino acidsmay be a non-natural amino acid); a reaction with two amino acids, eachof which may be naturally encoded or non-naturally encoded, with alinker, polymer, or other molecule under suitable conditions; etc.

In some embodiments, one or more amino acid substitutions in the insulinpolypeptide may be with one or more naturally occurring or non-naturallyencoded amino acids. In some embodiments the amino acid substitutions inthe insulin polypeptide may be with naturally occurring or non-naturallyencoded amino acids, provided that at least one substitution is with anon-naturally encoded amino acid. In some embodiments, one or more aminoacid substitutions in the insulin polypeptide may be with one or morenaturally occurring amino acids, and additionally at least onesubstitution is with a non-naturally encoded amino acid.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group, an acetyl group, an aminooxy group, a hydrazine group, ahydrazide group, a semicarbazide group, an azide group, or an alkynegroup.

In some embodiments, the non-naturally encoded amino acid comprises acarbonyl group. In some embodiments, the non-naturally encoded aminoacid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R2 is H, an alkyl, aryl, substituted alkyl, andsubstituted aryl; and R3 is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R4 is H, an amino acid, a polypeptide,or a carboxy terminus modification group.

In some embodiments, the non-naturally encoded amino acid comprises anaminooxy group. In some embodiments, the non-naturally encoded aminoacid comprises a hydrazide group. In some embodiments, the non-naturallyencoded amino acid comprises a hydrazine group. In some embodiments, thenon-naturally encoded amino acid residue comprises a semicarbazidegroup.

In some embodiments, the non-naturally encoded amino acid residuecomprises an azide group. In some embodiments, the non-naturally encodedamino acid has the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the non-naturally encoded amino acid comprises analkyne group. In some embodiments, the non-naturally encoded amino acidhas the structure:

wherein n is 0-10; R1 is an alkyl, aryl, substituted alkyl, orsubstituted aryl; X is O, N, S or not present; m is 0-10, R2 is H, anamino acid, a polypeptide, or an amino terminus modification group, andR3 is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, the polypeptide is an insulin polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonist. Insome embodiments, the insulin polypeptide agonist, partial agonist,antagonist, partial antagonist, or inverse agonist comprises anon-naturally encoded amino acid linked to a water soluble polymer. Insome embodiments, the water soluble polymer comprises a poly(ethyleneglycol) moiety. In some embodiments, the insulin polypeptide agonist,partial agonist, antagonist, partial antagonist, or inverse agonistcomprises a non-naturally encoded amino acid and one or morepost-translational modification, linker, polymer, or biologically activemolecule.

The present invention also provides isolated nucleic acids comprising apolynucleotide that hybridizes under stringent conditions nucleic acidsthat encode insulin polypeptides of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, and 12. The present invention also provides isolated nucleicacids comprising a polynucleotide that hybridizes under stringentconditions nucleic acids that encode insulin polypeptides of SEQ ID NOs:1 and 2. The present invention also provides isolated nucleic acidscomprising a polynucleotide or polynucleotides that hybridize understringent conditions to polynucleotides that encode polypeptides shownas SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 wherein thepolynucleotide comprises at least one selector codon. The presentinvention also provides isolated nucleic acids comprising apolynucleotide or polynucleotides that hybridize under stringentconditions to polynucleotides that encode polypeptides shown as SEQ IDNOs: 1 and 2 wherein the polynucleotide comprises at least one selectorcodon. The present invention provides isolated nucleic acids comprisinga polynucleotide that encodes the polypeptides shown as SEQ ID NOs.: 1and 2. The present invention also provides isolated nucleic acidscomprising a polynucleotide that encodes the polypeptides shown as SEQID NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. The presentinvention provides isolated nucleic acids comprising a polynucleotidethat encodes the polypeptides shown as SEQ ID NOs.: 1 and 2 with one ormore non-naturally encoded amino acids. The present invention alsoprovides isolated nucleic acids comprising a polynucleotide that encodesthe polypeptides shown as SEQ ID NOs.: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, and 12 with one or more non-naturally encoded amino acids. It isreadily apparent to those of ordinary skill in the art that a number ofdifferent polynucleotides can encode any polypeptide of the presentinvention.

In some embodiments, the selector codon is selected from the groupconsisting of an amber codon, ochre codon, opal codon, a unique codon, arare codon, a five-base codon, and a four-base codon.

The present invention also provides methods of making an insulinpolypeptide linked to a water soluble polymer. In some embodiments, themethod comprises contacting an isolated insulin polypeptide comprising anon-naturally encoded amino acid with a water soluble polymer comprisinga moiety that reacts with the non-naturally encoded amino acid. In someembodiments, the non-naturally encoded amino acid incorporated into theinsulin polypeptide is reactive toward a water soluble polymer that isotherwise unreactive toward any of the 20 common amino acids. In someembodiments, the non-naturally encoded amino acid incorporated into theinsulin polypeptide is reactive toward a linker, polymer, orbiologically active molecule that is otherwise unreactive toward any ofthe 20 common amino acids.

In some embodiments, the insulin polypeptide linked to the water solublepolymer is made by reacting an insulin polypeptide comprising acarbonyl-containing amino acid with a poly(ethylene glycol) moleculecomprising an aminooxy, hydrazine, hydrazide or semicarbazide group. Insome embodiments, the aminooxy, hydrazine, hydrazide or semicarbazidegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage. In some embodiments, the aminooxy, hydrazine, hydrazide orsemicarbazide group is linked to the poly(ethylene glycol) moleculethrough a carbamate linkage.

In some embodiments, the insulin polypeptide linked to the water solublepolymer is made by reacting a poly(ethylene glycol) molecule comprisinga carbonyl group with a polypeptide comprising a non-naturally encodedamino acid that comprises an aminooxy, hydrazine, hydrazide orsemicarbazide group.

In some embodiments, the insulin polypeptide linked to the water solublepolymer is made by reacting a insulin polypeptide comprising analkyne-containing amino acid with a poly(ethylene glycol) moleculecomprising an azide moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the insulin polypeptide linked to the water solublepolymer is made by reacting a insulin polypeptide comprising anazide-containing amino acid with a poly(ethylene glycol) moleculecomprising an alkyne moiety. In some embodiments, the azide or alkynegroup is linked to the poly(ethylene glycol) molecule through an amidelinkage.

In some embodiments, the poly(ethylene glycol) molecule has a molecularweight of between about 0.1 kDa and about 100 kDa. In some embodiments,the poly(ethylene glycol) molecule has a molecular weight of between 0.1kDa and 50 kDa.

In some embodiments, the poly(ethylene glycol) molecule is a branchedpolymer. In some embodiments, each branch of the poly(ethylene glycol)branched polymer has a molecular weight of between 1 kDa and 100 kDa, orbetween 1 kDa and 50 kDa.

In some embodiments, the water soluble polymer linked to the insulinpolypeptide comprises a polyalkylene glycol moiety. In some embodiments,the non-naturally encoded amino acid residue incorporated into theinsulin polypeptide comprises a carbonyl group, an aminooxy group, ahydrazide group, a hydrazine, a semicarbazide group, an azide group, oran alkyne group. In some embodiments, the non-naturally encoded aminoacid residue incorporated into the insulin polypeptide comprises acarbonyl moiety and the water soluble polymer comprises an aminooxy,hydrazide, hydrazine, or semicarbazide moiety. In some embodiments, thenon-naturally encoded amino acid residue incorporated into the insulinpolypeptide comprises an alkyne moiety and the water soluble polymercomprises an azide moiety. In some embodiments, the non-naturallyencoded amino acid residue incorporated into the insulin polypeptidecomprises an azide moiety and the water soluble polymer comprises analkyne moiety.

The present invention also provides compositions comprising an insulinpolypeptide comprising a non-naturally encoded amino acid and apharmaceutically acceptable carrier. In some embodiments, thenon-naturally encoded amino acid is linked to a water soluble polymer.

The present invention also provides cells comprising a polynucleotideencoding the insulin polypeptide comprising a selector codon. In someembodiments, the cells comprise an orthogonal RNA synthetase and/or anorthogonal tRNA for substituting a non-naturally encoded amino acid intothe insulin polypeptide.

The present invention also provides methods of making an insulinpolypeptide comprising a non-naturally encoded amino acid. In someembodiments, the methods comprise culturing cells comprising apolynucleotide or polynucleotides encoding an insulin polypeptide, anorthogonal RNA synthetase and/or an orthogonal tRNA under conditions topermit expression of the insulin polypeptide; and purifying the insulinpolypeptide from the cells and/or culture medium.

The present invention also provides methods of increasing therapeutichalf-life, serum half-life or circulation time of insulin polypeptides.The present invention also provides methods of modulating immunogenicityof insulin polypeptides. In some embodiments, the methods comprisesubstituting a non-naturally encoded amino acid for any one or moreamino acids in naturally occurring insulin polypeptides and/or linkingthe insulin polypeptide to a linker, a polymer, a water soluble polymer,or a biologically active molecule.

The present invention also provides methods of treating a patient inneed of such treatment with an effective amount of an insulin moleculeof the present invention. In some embodiments, the methods compriseadministering to the patient a therapeutically-effective amount of apharmaceutical composition comprising an insulin polypeptide comprisinga non-naturally-encoded amino acid and a pharmaceutically acceptablecarrier. In some embodiments, the non-naturally encoded amino acid islinked to a water soluble polymer. In some embodiments, the insulinpolypeptide is glycosylated. In some embodiments, the insulinpolypeptide is not glycosylated.

The present invention also provides insulin polypeptides comprising asequence shown in SEQ ID NO: 1 and 2, or any other insulin polypeptidesequence (a non-limiting example of these would be SEQ ID NOs: 3 through12) except that at least one amino acid is substituted by anon-naturally encoded amino acid. In some embodiments, the non-naturallyencoded amino acid is linked to a water soluble polymer. In someembodiments, the water soluble polymer comprises a poly(ethylene glycol)moiety. In some embodiments, the non-naturally encoded amino acidcomprises a carbonyl group, an aminooxy group, a hydrazide group, ahydrazine group, a semicarbazide group, an azide group, or an alkynegroup.

The present invention also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and an insulinpolypeptide comprising the sequence shown in SEQ ID NOs: 1 through 12,or any other insulin polypeptide sequence, wherein at least one aminoacid is substituted by a non-naturally encoded amino acid. The presentinvention also provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and an insulin polypeptidecomprising an A and B chain (e.g. SEQ ID NOs: 1 and 2 would makeinsulin, SEQ ID NOs 3 and 4 would make insulin lispro, etc.) thesequence shown in SEQ ID NOs: 1 through 12, or any other insulinpolypeptide sequence, wherein at least one amino acid is substituted bya non-naturally encoded amino acid. The present invention also providespharmaceutical compositions comprising a pharmaceutically acceptablecarrier and an insulin polypeptide comprising the sequence shown in SEQID NO: 1 and 2. The present invention also provides pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and aninsulin polypeptide comprising the sequence shown in SEQ ID NO: 1through 12. In some embodiments, the non-naturally encoded amino acidcomprises a saccharide moiety. In some embodiments, the water solublepolymer is linked to the polypeptide via a saccharide moiety. In someembodiments, a linker, polymer, or biologically active molecule islinked to the insulin polypeptide via a saccharide moiety.

The present invention also provides an insulin polypeptide comprising awater soluble polymer linked by a covalent bond to the insulinpolypeptide at a single amino acid. In some embodiments, the watersoluble polymer comprises a poly(ethylene glycol) moiety. In someembodiments, the amino acid covalently linked to the water solublepolymer is a non-naturally encoded amino acid present in thepolypeptide.

The present invention provides an insulin polypeptide comprising atleast one linker, polymer, or biologically active molecule, wherein saidlinker, polymer, or biologically active molecule is attached to thepolypeptide through a functional group of a non-naturally encoded aminoacid ribosomally incorporated into the polypeptide. In some embodiments,the polypeptide is monoPEGylated. The present invention also provides aninsulin polypeptide comprising a linker, polymer, or biologically activemolecule that is attached to one or more non-naturally encoded aminoacid wherein said non-naturally encoded amino acid is ribosomallyincorporated into the polypeptide at pre-selected sites.

Included within the scope of this invention is the insulin leader orsignal sequence an example of which can be seen as proinulin. Theheterologous leader or signal sequence selected should be one that isrecognized and processed, e.g. by host cell secretion system to secreteand possibly cleaved by a signal peptidase, by the host cell. A methodof treating a condition or disorder with insulin or an insulinpolypeptide or analog of the present invention is meant to implytreating with insulin with or without a signal or leader peptide.

The present invention also provides methods of inducing an increase inglucose metabolism, said method comprising administering insulin to saidcells in an amount effective to induce an increase in glucose metabolicactivity.

In another embodiment, conjugation of the insulin polypeptide comprisingone or more non-naturally encoded amino acids to another molecule,including but not limited to PEG, provides substantially purifiedinsulin due to the unique chemical reaction utilized for conjugation tothe non-natural amino acid. Conjugation of insulin comprising one ormore non-naturally encoded amino acids to another molecule, such as PEG,may be performed with other purification techniques performed prior toor following the conjugation step to provide substantially pure insulin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows two models of the crystal structure of insulin are shownalong with the amino acid sequence of insulin.

FIG. 2 is a model of the crystal structure of insulin is shown. Selectedsites for substitution with a non-naturally encoded amino acid areshown.

FIG. 3 is a drawing of primary proinsulin.

FIG. 4 shows drawings of insulin, humalog, novolog, glargine, anddetemir.

FIG. 5 shows the proinsulin expression plasmid pVK6-LisPro Insulin aswell as the sequence of the LisPro Insulin insert.

FIG. 6 shows an SDS-PAGE gel analysis with insulin polypeptides of thepresent invention include those substituted at Q15 A chain (SEQ ID NO:3), Y14 A chain (SEQ ID NO: 3), R22 B chain (SEQ ID NO: 4), F1 B chain(SEQ ID NO: 4), G1 A chain (SEQ ID NO: 3), S9 A chain (SEQ ID NO: 3),and K28 B chain (SEQ ID NO: 4), each substitution made with anon-natural amino acid (pAF).

FIG. 7 shows an SEC-HPLC of PEGylated and unPEGylated insulinpolypeptides.

FIG. 8 shows a diagram of a plasmid used for cloning into Pichia.

FIG. 9 shows an Area Under the Curve bar graph for two insulinpolypeptides of the present invention, A14pAF-PEG-A21N andA14pAF-PEG-A21G.

FIG. 10 shows a gel of the results from the refolding reaction for A21Ginsulin polypeptide, as discuss further in Example 36.

FIG. 11 shows an HPLC of QHP purified and refolded proinsulin fromExample 36.

FIG. 12 shows an HPLC of the PEGylation reaction described in Example36.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “insulin” or “insulinpolypeptide” and various hyphenated and unhyphenated forms is areference to one or more such proteins and includes equivalents thereofknown to those of ordinary skill in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

The term “substantially purified” refers to an insulin polypeptide thatmay be substantially or essentially free of components that normallyaccompany or interact with the protein as found in its naturallyoccurring environment, i.e. a native cell, or host cell in the case ofrecombinantly produced insulin polypeptides. Insulin polypeptide thatmay be substantially free of cellular material includes preparations ofprotein having less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 4%, less than about 3%, less than about 2%, or less thanabout 1% (by dry weight) of contaminating protein. When the insulinpolypeptide or variant thereof is recombinantly produced by the hostcells, the protein may be present at about 30%, about 25%, about 20%,about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about1% or less of the dry weight of the cells. When the insulin polypeptideor variant thereof is recombinantly produced by the host cells, theprotein may be present in the culture medium at about 5 g/L, about 4g/L, about 3 g/L, about 2 g/L, about 1 g/L, about 750 mg/L, about 500mg/L, about 250 mg/L, about 100 mg/L, about 50 mg/L, about 10 mg/L, orabout 1 mg/L or less of the thy weight of the cells. Thus,“substantially purified” insulin polypeptide as produced by the methodsof the present invention may have a purity level of at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, specifically, a purity level of at least about 75%,80%, 85%, and more specifically, a purity level of at least about 90%, apurity level of at least about 95%, a purity level of at least about 99%or greater as determined by appropriate methods such as SDS/PAGEanalysis, RP-HPLC, SEC, and capillary electrophoresis.

A “recombinant host cell” or “host cell” refers to a cell that includesan exogenous polynucleotide, regardless of the method used forinsertion, for example, direct uptake, transduction, f-mating, or othermethods known in the art to create recombinant host cells. The exogenouspolynucleotide may be maintained as a nonintegrated vector, for example,a plasmid, or alternatively, may be integrated into the host genome.

As used herein, the term “medium” or “media” includes any culturemedium, solution, solid, semi-solid, or rigid support that may supportor contain any host cell, including bacterial host cells, yeast hostcells, insect host cells, plant host cells, eukaryotic host cells,mammalian host cells, CHO cells, prokaryotic host cells, E. coli, orPseudomonas host cells, and cell contents. Thus, the term may encompassmedium in which the host cell has been grown, e.g., medium into whichthe insulin polypeptide has been secreted, including medium eitherbefore or after a proliferation step. The term also may encompassbuffers or reagents that contain host cell lysates, such as in the casewhere the insulin polypeptide is produced intracellularly and the hostcells are lysed or disrupted to release the insulin polypeptide.

“Reducing agent,” as used herein with respect to protein refolding, isdefined as any compound or material which maintains sulfhydryl groups inthe reduced state and reduces intra- or intermolecular disulfide bonds.Suitable reducing agents include, but are not limited to, dithiothreitol(DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine(2-aminoethanethiol), and reduced glutathione. It is readily apparent tothose of ordinary skill in the art that a wide variety of reducingagents are suitable for use in the methods and compositions of thepresent invention.

“Oxidizing agent,” as used hereinwith respect to protein refolding, isdefined as any compound or material which is capable of removing anelectron from a compound being oxidized. Suitable oxidizing agentsinclude, but are not limited to, oxidized glutathione, cystine,cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. Itis readily apparent to those of ordinary skill in the art that a widevariety of oxidizing agents are suitable for use in the methods of thepresent invention.

“Denaturing agent” or “denaturant,” as used herein, is defined as anycompound or material which will cause a reversible unfolding of aprotein. The strength of a denaturing agent or denaturant will bedetermined both by the properties and the concentration of theparticular denaturing agent or denaturant. Suitable denaturing agents ordenaturants may be chaotropes, detergents, organic solvents, watermiscible solvents, phospholipids, or a combination of two or more suchagents. Suitable chaotropes include, but are not limited to, urea,guanidine, and sodium thiocyanate. Useful detergents may include, butare not limited to, strong detergents such as sodium dodecyl sulfate, orpolyoxyethylene ethers (e.g. Tween or Triton detergents), Sarkosyl, mildnon-ionic detergents (e.g., digitonin), mild cationic detergents such asN->2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents(e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergentsincluding, but not limited to, sulfobetaines (Zwittergent),3-(3-chlolamidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and3-(3-chlolamidopropyl)dimethylammonio-2-hydroxy-1-propane sultanate(CHAPSO). Organic, water miscible solvents such as acetonitrile, loweralkanols (especially C2-C4 alkanols such as ethanol or isopropanol), orlower alkandiols (especially C2-C4 alkandiols such as ethylene-glycol)may be used as denaturants. Phospholipids useful in the presentinvention may be naturally occurring phospholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, andphosphatidylinositol or synthetic phospholipid derivatives or variantssuch as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

“Refolding,” as used herein describes any process, reaction or methodwhich transforms disulfide bond containing polypeptides from animproperly folded or unfolded state to a native or properly foldedconformation with respect to disulfide bonds.

“Cofolding,” as used herein, refers specifically to refolding processes,reactions, or methods which employ at least two polypeptides whichinteract with each other and result in the transformation of unfolded orimproperly folded polypeptides to native, properly folded polypeptides.

The term “proinsulin” as used herein is a properly cross-line protein ofthe formula: B-C-A

wherein:A is the A chain of insulin or a functional derivative thereof;B is the B chain of insulin or a functional derivative thereof having an.epsilon.-amino group; andC is the connecting peptide of proinsulin. Preferably, proinsulin is theA chain of human insulin, the B chain of human insulin, and C is thenatural connecting peptide. When proinsulin is the natural sequence,proinsulin possesses three free amino groups: Phenylalanine(1)(.alpha.-amino group), Lysine(29) (.epsilon.-amino group) and Lysine(64)(.epsilon.-amino group).

The term “insulin analog” as used herein is a properly cross-linedprotein exhibiting insulin activity of the formula:

A-B

wherein:A is the A chain of insulin or a functional derivative of the insulin Achain; andB is the B chain of insulin or a functional derivative of the insulin Bchain having an .epsilon.-amino group and at least one of A or Bcontains an amino acid modification from the natural sequence.

In the present specification, whenever the term insulin is used in aplural or a generic sense it is intended to encompass both naturallyoccurring insulins and insulin analogues and derivatives thereof. By“insulin polypeptide” as used herein is meant a compound having amolecular structure similar to that of human insulin including thedisulfide bridges between Cys.sup.A7 and Cys.sup.B7 and betweenCys.sup.A20 and Cys.sup.B19 and an internal disulfide bridge betweenCys.sup.A6 and Cys.sup.A11, and which have insulin activity.

The term “insulin” as used herein, refers to human insulin, whose aminoacid sequence and spatial structure are well-known. Human insulin iscomprised of a twenty-one amino acid A-chain and a thirty amino acidB-chain which are cross-linked by disulfide bonds. A properlycross-linked insulin contains three disulfide bridges: one betweenposition 7 of the A-chain and position 7 of the B-chain, a secondbetween position 20 of the A-chain and position 19 of the B-chain, and athird between positions 6 and 11 of the A-chain [Nicol, D. S. H. W. andSmith, L. F., Nature, 187, 483-485 (1960)].

Insulin peptides including, but not limited to, insulin, human; insulin,porcine; IGF-I, human; insulin-like growth factor II (69-84);pro-insulin-like growth factor II (68-102), human; pro-insulin-likegrowth factor II (105-128), human; [AspB28]-insulin, human;[LysB28]-insulin, human; [LeuB28]-insulin, human; [ValB28]-insulin,human; [AlaB28]-insulin, human; [AspB28, ProB29]-insulin, human;[LysB28, ProB29]-insulin, human; [LeuB28, ProB29]-insulin, human;[ValB28, ProB29]-insulin, human; [AlaB28, ProB29]-insulin, human;[GlyA21]-insulin, human; [GlyA21 GlnB3]-insulin, human;[AlaA21]-insulin, human; [AlaA21 Gln.sup.B3] insulin, human;[GlnB3]-insulin, human; [GlnB30]-insulin, human; [GlyA21GluB30]-insulin, human; [GlyA21 GlnB3 GluB30]-insulin, human; [GlnB3GluB30]-insulin, human; B22-B30 insulin, human; B23-B30 insulin, human;B25-B30 insulin, human; B26-B30 insulin, human; B27-B30 insulin, human;B29-B30 human; the A chain of human insulin, and the B chain of humaninsulin.

The term “insulin analog” means a protein that has an A-chain and aB-chain that have substantially the same amino acid sequences as theA-chain and/or B-chain of human insulin, respectively, but differ fromthe A-chain and B-chain of human insulin by having one or more aminoacid deletions, one or more amino acid replacements, and/or one or moreamino acid additions that do not destroy the insulin activity of theinsulin analog. An insulin analog having an isoelectric point that is“higher than” the isoelectric point of insulin is one type of insulinanalog. Another type of insulin analog is a “monomeric insulin analog.”

A “monomeric insulin analog” is a fast-acting analog of human insulin,including, for example, human insulin wherein Pro at position B28 issubstituted with Asp, Lys, Leu, Val, or Ala, and wherein Lys at positionB29 is Lys or is substituted with Pro. Another monomeric insulin analog,also known as des(B27) human insulin, is human insulin wherein Thr atposition 27 of the B-chain is deleted. Monomeric insulin analogs aredisclosed in Chance, R. E., et al., U.S. Pat. No. 5,514,646, issued May7, 1996; Brems, D. N., et al. Protein Engineering, 5, 527-533 (1992);Brange, J. J. V., et al., EPO publication No. 214,826 (published Mar.18, 1987); and Brange, J. J. V., et al., Current Opinion in StructuralBiology, 1, 934-940 (1991). The monomeric insulin analogs employed inthe present formulations are properly cross-linked at the same positionsas in human insulin.

Insulin peptides including, but not limited to, insulin, human; insulin,porcine; IGF-I, human; insulin-like growth factor II (69-84);pro-insulin-like growth factor II (68-102), human; pro-insulin-likegrowth factor II (105-128), human; [AspB28]-insulin, human;[LysB28]-insulin, human; [LeuB28]-insulin, human; [ValB28]-insulin,human; [AlaB28]-insulin, human; [AspB28, ProB29]-insulin, human;[LysB28, ProB29]-insulin, human; [LeuB28, ProB29]-insulin, human;[ValB28, ProB29]-insulin, human; [AlaB28, ProB29]-insulin, human;[GlyA21]-insulin, human; [GlyA21 GlnB3]-insulin, human;[AlaA21]-insulin, human; [AlaA21 Gln.sup.B3] insulin, human;[GlnB3]-insulin, human; [GlnB30]-insulin, human; [GlyA21GluB30]-insulin, human; [GlyA21 GlnB3 GluB30]-insulin, human; [GlnB3GluB30]-insulin, human; B22-B30 insulin, human; B23-B30 insulin, human;B25-830 insulin, human; B26-B30 insulin, human; B27-B30 insulin, human;B29-B30 human; the A chain of human insulin, and the B chain of humaninsulin.

In a further aspect, the invention provides recombinant nucleic acidsencoding the variant proteins, expression vectors containing the variantnucleic acids, host cells comprising the variant nucleic acids and/orexpression vectors, and methods for producing the variant proteins. Inan additional aspect, the invention provides treating an insulinresponsive disorder by administering to a patient a variant protein,usually with a pharmaceutical carrier, in a therapeutically effectiveamount. In a further aspect, the invention provides methods formodulating immunogenicity (particularly reducing immunogenicity) ofinsulin polypeptides by altering MHC Class II epitopes.

The term “insulin polypeptide” also includes the pharmaceuticallyacceptable salts and prodrugs, and prodrugs of the salts, polymorphs,hydrates, solvates, biologically-active fragments, biologically activevariants and stereoisomers of the naturally-occurring insulin as well asagonist, mimetic, and antagonist variants of the naturally-occurringinsulin and polypeptide fusions thereof. Fusions comprising additionalamino acids at the amino terminus, carboxyl terminus, or both, areencompassed by the term “insulin polypeptide.” Exemplary fusionsinclude, but are not limited to, e.g., methionyl insulin in which amethionine is linked to the N-terminus of insulin resulting from therecombinant expression of the mature form of insulin lacking the leaderor signal peptide or portion thereof (a methionine is linked to theN-terminus of insulin resulting from the recombinant expression),fusions for the purpose of purification (including, but not limited to,to poly-histidine or affinity epitopes), fusions with serum albuminbinding peptides and fusions with serum proteins such as serum albumin.U.S. Pat. No. 5,750,373, which is incorporated by reference herein,describes a method for selecting novel proteins such as growth hormoneand antibody fragment variants having altered binding properties fortheir respective receptor molecules. The method comprises fusing a geneencoding a protein of interest to the carboxy terminal domain of thegene III coat protein of the filamentous phage M13. Chimeric moleculescomprising insulin and one or more other molecules. The chimericmolecule can contain specific regions or fragments of one or both of theinsulin and the other molecule(s). Any such fragments can be preparedfrom the proteins by standard biochemical methods, or by expressing apolynucleotide encoding the fragment. Insulin, or a fragment thereof,can be produced as a fusion protein comprising human serum albumin(HSA), Fc, or a portion thereof. Such fusion constructs are suitable forenhancing expression of the insulin, or fragment thereof, in aneukaryotic host cell. Exemplary USA portions include the N-terminalpolypeptide (amino acids 1-369, 1-419, and intermediate lengths startingwith amino acid 1), as disclosed in U.S. Pat. No. 5,766,883, andpublication WO 97/24445, which are incorporated by reference herein.Other chimeric polypeptides can include a HSA protein with insulin, orfragments thereof, attached to each of the C-terminal and N-terminalends of the USA. Such USA constructs are disclosed in U.S. Pat. No.5,876,969, which is incorporated by reference herein. Other fusions maybe created by fusion of insulin with a) the Fc portion of animmunoglobulin; b) an analog of the Fc portion of an immunoglobulin; andc) fragments of the Fc portion of an immunoglobulin.

Various references disclose modification of polypeptides by polymerconjugation or glycosylation. The term “insulin polypeptide” includespolypeptides conjugated to a polymer such as PEG and may be comprised ofone or more additional derivitizations of cysteine, lysine, or otherresidues. In addition, the insulin polypeptide may comprise a linker orpolymer, wherein the amino acid to which the linker or polymer isconjugated may be a non-natural amino acid according to the presentinvention, or may be conjugated to a naturally encoded amino acidutilizing techniques known in the art such as coupling to lysine orcysteine.

The term “insulin polypeptide” also includes glycosylated insulin, suchas but not limited to, polypeptides glycosylated at any amino acidposition, N-linked or O-linked glycosylated forms of the polypeptide.Variants containing single nucleotide changes are also considered asbiologically active variants of insulin polypeptide. In addition, splicevariants are also included. The term “insulin polypeptide” also includesinsulin polypeptide heterodimers, homodimers, heteromultimers, orhomomultimers of any one or more insulin polypeptides or any otherpolypeptide, protein, carbohydrate, polymer, small molecule, linker,ligand, or other biologically active molecule of any type, linked bychemical means or expressed as a fusion protein, as well as polypeptideanalogues containing, for example, specific deletions or othermodifications yet maintain biological activity.

The term “insulin polypeptide” or “insulin” encompasses insulinpolypeptides comprising one or more amino acid substitutions, additionsor deletions. Insulin polypeptides of the present invention may becomprised of modifications with one or more natural amino acids inconjunction with one or more non-natural amino acid modification.Exemplary substitutions in a wide variety of amino acid positions innaturally-occurring insulin polypeptides have been described, includingbut not limited to substitutions that modulate pharmaceutical stability,that modulate one or more of the biological activities of the insulinpolypeptide, such as but not limited to, increase agonist activity,increase solubility of the polypeptide, decrease proteasesusceptibility, convert the polypeptide into an antagonist, etc. and areencompassed by the term “insulin polypeptide.” In some embodiments, theinsulin antagonist comprises a non-naturally encoded amino acid linkedto a water soluble polymer that is present in a receptor binding regionof the insulin molecule.

In some embodiments, the insulin polypeptides further comprise anaddition, substitution or deletion that modulates biological activity ofthe insulin polypeptide. In some embodiments, the insulin polypeptidesfurther comprise an addition, substitution or deletion that modulatesanti-viral activity of the insulin polypeptide. In some embodiments, theinsulin polypeptides further comprise an addition, substitution ordeletion that enhances anti-viral activity of the insulin polypeptide.For example, the additions, substitutions or deletions may modulate oneor more properties or activities of insulin. For example, the additions,substitutions or deletions may modulate affinity for the insulinreceptor, modulate circulating half-life, modulate therapeutichalf-life, modulate stability of the polypeptide, modulate cleavage byproteases, modulate dose, modulate release or bio-availability,facilitate purification, or improve or alter a particular route ofadministration. Similarly, insulin polypeptides may comprise proteasecleavage sequences, reactive groups, antibody-binding domains (includingbut not limited to, FLAG or poly-His) or other affinity based sequences(including but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including but not limited to, biotin) that improve detection(including but not limited to, GFP), purification or other traits of thepolypeptide.

The term “insulin polypeptide” also encompasses homodimers,heterodimers, homomultimers, and heteromultimers that are linked,including but not limited to those linked directly via non-naturallyencoded amino acid side chains, either to the same or differentnon-naturally encoded amino acid side chains, to naturally-encoded aminoacid side chains, or indirectly via a linker. Exemplary linkersincluding but are not limited to, small organic compounds, water solublepolymers of a variety of lengths such as poly(ethylene glycol) orpolydextran, or polypeptides of various lengths.

A “non-naturally encoded amino acid” refers to an amino acid that is notone of the 20 common amino acids or pyrrolysine or selenocysteine. Otherterms that may be used synonymously with the term “non-naturally encodedamino acid” are “non-natural amino acid,” “unnatural amino acid,”“non-naturally-occurring amino acid,” and variously hyphenated andnon-hyphenated versions thereof. The term “non-naturally encoded aminoacid” also includes, but is not limited to, amino acids that occur bymodification (e.g. post-translational modifications) of a naturallyencoded amino acid (including but not limited to, the 20 common aminoacids or pyrrolysine and selenocysteine) but are not themselvesnaturally incorporated into a growing polypeptide chain by thetranslation complex. Examples of such non-naturally-occurring aminoacids include, but are not limited to, N-acetylglucosaminyl-L-serine,N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

An “amino terminus modification group” refers to any molecule that canbe attached to the amino terminus of a polypeptide. Similarly, a“carboxy terminus modification group” refers to any molecule that can beattached to the carboxy terminus of a polypeptide. Terminus modificationgroups include, but are not limited to, various water soluble polymers,peptides or proteins such as serum albumin, or other moieties thatincrease serum half-life of peptides.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “linkage” or “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide.

The term “biologically active molecule”, “biologically active moiety” or“biologically active agent” when used herein means any substance whichcan affect any physical or biochemical properties of a biologicalsystem, pathway, molecule, or interaction relating to an organism,including but not limited to, viruses, bacteria, bacteriophage,transposon, prion, insects, fungi, plants, animals, and humans. Inparticular, as used herein, biologically active molecules include, butare not limited to, any substance intended for diagnosis, cure,mitigation, treatment, or prevention of disease in humans or otheranimals, or to otherwise enhance physical or mental well-being of humansor animals. Examples of biologically active molecules include, but arenot limited to, peptides, proteins, enzymes, small molecule drugs,vaccines, immunogens, hard drugs, soft drugs, carbohydrates, inorganicatoms or molecules, dyes, lipids, nucleosides, radionuclides,oligonucleotides, toxoids, toxins, prokaryotic and eukaryotic cells,viruses, polysaccharides, nucleic acids and portions thereof obtained orderived from viruses, bacteria, insects, animals or any other cell orcell type, liposomes, microparticles and micelles. The insulinpolypeptides may be added in a micellular formulation; see U.S. Pat. No.5,833,948, which is incorporated by reference herein in its entirety.Classes of biologically active agents that are suitable for use with theinvention include, but are not limited to, drugs, prodrugs,radionuclides, imaging agents, polymers, antibiotics, fungicides,anti-viral agents, anti-inflammatory agents, anti-tumor agents,cardiovascular agents, anti-anxiety agents, hormones, growth factors,steroidal agents, microbially derived toxins, and the like.

A “bifunctional polymer” refers to a polymer comprising two discretefunctional groups that are capable of reacting specifically with othermoieties (including but not limited to, amino acid side groups) to formcovalent or non-covalent linkages. A bifunctional linker having onefunctional group reactive with a group on a particular biologicallyactive component, and another group reactive with a group on a secondbiological component, may be used to form a conjugate that includes thefirst biologically active component, the bifunctional linker and thesecond biologically active component. Many procedures and linkermolecules for attachment of various compounds to peptides are known.See, e.g., European Patent Application No. 188,256; U.S. Pat. Nos.4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789which are incorporated by reference herein. A “multi-functional polymer”refers to a polymer comprising two or more discrete functional groupsthat are capable of reacting specifically with other moieties (includingbut not limited to, amino acid side groups) to form covalent ornon-covalent linkages. A bi-functional polymer or multi-functionalpolymer may be any desired length or molecular weight, and may beselected to provide a particular desired spacing or conformation betweenone or more molecules linked to the insulin and its receptor or insulin.

Were substituent groups are specified by their conventional chemicalformulas, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, for example, the structure CH2O isequivalent to the structure —OCH2.

The term “substituents” includes but is not limited to “non-interferingsubstituents”. “Non-interfering substituents” are those groups thatyield stable compounds. Suitable non-interfering substituents orradicals include, but are not limited to, halo, C1-C10 alkyl, C2-C10alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C1-C12 aralkyl, C1-C12 alkaryl,C3-C12 cycloalkyl, C3-C12 cycloalkenyl, phenyl, substituted phenyl,toluoyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C2-C12 alkoxyaryl,C7-C12 aryloxyalkyl, C7-C12 oxyaryl, C1-C6 alkylsulfinyl, C1-C10alkylsulfonyl, —(CH2)m —O—(C1-C10 alkyl) wherein m is from 1 to 8, aryl,substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical,substituted heterocyclic radical, nitroalkyl, —NO2, —CN, —NRC(O)—(C1-C10alkyl), —C(O)—(C1-C10 alkyl), C2-C10 alkyl thioalkyl, —C(O)O—(C1-C10alkyl), —OH, —SO2, ═S, —COOH, —NR2, carbonyl, —C(O)—(C1-C10 alkyl)-CF3,—C(O)—CF3, —C(O)NR2, —(C1-C10 aryl)-S—(C6-C10 aryl), —C(O)—(C1-C10aryl), —(CH2)m-O—)C1-C10 alkyl) wherein each m is from 1 to 8, —C(O)NR2,—C(S)NR2, —SO2NR2, —NRC(O)NR2, —NRC(S) NR2, salts thereof, and the like.Each R as used herein is H, alkyl or substituted alkyl, aryl orsubstituted aryl, aralkyl, or alkaryl.

The term “halogen” includes fluorine, chlorine, iodine, and bromine.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e. C1-C10means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butyryl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by the structures —CH2CH2- and —CH2CH2CH2CH2-, and furtherincludes those groups described below as “heteroalkylene.” Typically, analkyl (or alkylene) group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being a particularembodiment of the methods and compositions described herein. A “loweralkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen andsulfur atoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) O, N and S and Si may beplaced at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH2-CH2-O—CH3,—CH2-CH2-NH—CH3, —CH2-CH2-N(CH3)-CH3, —CH2-S—CH2-CH3, —CH2-CH2,—S(O)—CH3, —CH2-CH2-S(O)2-CH3, —CH═CH—O—CH3, —Si(CH3)3, —CH2-CH═N—OCH3,and —CH═CH—N(CH3)-—CH3. Up to two heteroatoms may be consecutive, suchas, for example, —CH2-NH—OCH3 and —CH2-O—Si(CH3)3. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH2-CH2-S—CH2CH2- and —CH2-S—CH2-CH2-NH—CH2-. Forheteroalkylene groups, the same or different heteroatoms can also occupyeither or both of the chain termini (including but not limited to,alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino,aminooxyalkylene, and the like). Still further, for alkylene andheteroalkylene linking groups, no orientation of the linking group isimplied by the direction in which the formula of the linking group iswritten. For example, the formula —C(O)2R′ represents both —C(O)2R′ and—R′C(O)2.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkylor heterocycloalkyl include saturated, partially unsaturated and fullyunsaturated ring linkages. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkylinclude, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl),1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl,3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl,tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl,2-piperazinyl, and the like. Additionally, the term encompasses bicyclicand tricyclic ring structures. Similarly, the term “heterocycloalkylene”by itself or as part of another substituent means a divalent radicalderived from heterocycloalkyl, and the term “cycloalkylene” by itself oras part of another substituent means a divalent radical derived fromcycloalkyl.

As used herein, the term “water soluble polymer” refers to any polymerthat is soluble in aqueous solvents. Linkage of water soluble polymersto insulin polypeptides can result in changes including, but not limitedto, increased or modulated serum half-life, or increased or modulatedtherapeutic half-life relative to the unmodified form, modulatedimmunogenicity, modulated physical association characteristics such asaggregation and multimer formation, altered receptor binding, alteredbinding to one or more binding partners, and altered receptordimerization or multimerization. The water soluble polymer may or maynot have its own biological activity, and may be utilized as a linkerfor attaching insulin to other substances, including but not limited toone or more insulin polypeptides, or one or more biologically activemolecules. Suitable polymers include, but are not limited to,polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C10alkoxy or aryloxy derivatives thereof (described in U.S. Pat. No.5,252,714 which is incorporated by reference herein),monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinylalcohol, polyamino acids, divinylether maleic anhydride,N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivativesincluding dextran sulfate, polypropylene glycol, polypropyleneoxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin,heparin fragments, polysaccharides, oligosaccharides, glycans, celluloseand cellulose derivatives, including but not limited to methylcelluloseand carboxymethyl cellulose, starch and starch derivatives,polypeptides, polyalkylene glycol and derivatives thereof, copolymers ofpolyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers,and alpha-beta-poly[(2-hydroxyethyl)-DL-aspartamide, and the like, ormixtures thereof. Examples of such water soluble polymers include, butare not limited to, polyethylene glycol and serum albumin.

As used herein, the term “polyalkylene glycol” or “poly(alkene glycol)”refers to polyethylene glycol (poly(ethylene glycol)), polypropyleneglycol, polybutylene glycol, and derivatives thereof. The term“polyalkylene glycol” encompasses both linear and branched polymers andaverage molecular weights of between 0.1 kDa and 100 kDa. Otherexemplary embodiments are listed, for example, in commercial suppliercatalogs, such as Shearwater Corporation's catalog “Polyethylene Glycoland Derivatives for Biomedical Applications” (2001).

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent which can be a single ring or multiplerings (including but not limited to, from 1 to 3 rings) which are fusedtogether or linked covalently. The term “heteroaryl” refers to arylgroups (or rings) that contain from one to four heteroatoms selectedfrom N, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. Aheteroaryl group can be attached to the remainder of the moleculethrough a heteroatom. Non-limiting examples of aryl and heteroarylgroups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl,pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl,3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl,3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl,purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl,2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituentsfor each of the above noted aryl and heteroaryl ring systems areselected from the group of acceptable substituents described below.

For brevity, the term “aryl” when used in combination with other terms(including but not limited to, aryloxy, arylthioxy, arylalkyl) includesboth aryl and heteroaryl rings as defined above. Thus, the term“arylalkyl” is meant to include those radicals in which an aryl group isattached to an alkyl group (including but not limited to, benzyl,phenethyl, pyridylmethyl and the like) including those alkyl groups inwhich a carbon atom (including but not limited to, a methylene group)has been replaced by, for example, an oxygen atom (including but notlimited to, phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl,and the like).

Each of the above terms (including but not limited to, “alkyl,”“heteroalkyl,” “aryl” and “heteroaryl”) are meant to include bothsubstituted and unsubstituted forms of the indicated radical. Exemplarysubstituents for each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocyeloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′,-halogen, —SiR′R″R′″, OC(O)R′, —C(O)R′, —CO2R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, NR′ C(O)NR″R′″, —NR″C(O)2R′, —NR—C(NR′R″R′″)═NR″″,NRC(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″, NRSO2R′, —CN and —NO2in a number ranging from zero to (2m′+1), where m′ is the total numberof carbon atoms in such a radical. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF3 and —CH2CF3) and acyl (including but not limited to,—C(O)CH3, —C(O)CF3, —C(O)CH2OCH3, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, but are not limited to: halogen, OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, OC(O)R′, —C(O)R′, CO2R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, NR′ C(O)NR″R′″, —NR″C(O)2R′,NR—C(NR′R″R′″)═NR″″, NR C(NR′R″)═NR′″, —S(O)R′, —S(O)2R′, —S(O)2NR′R″,NRSO2R′, —CN and —NO2, —R′, —N3, —CH(Ph)2, fluoro(C1-C4)alkoxy, andfluoro(C1-C4)alkyl, in a number ranging from zero to the total number ofopen valences on the aromatic ring system; and where R′, R″, R′″ and R″″are independently selected from hydrogen, alkyl, heteroalkyl, aryl andheteroaryl. When a compound of the invention includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″ and R″″ groups when more than one of these groupsis present.

As used herein, the term “modulated serum half-life” means the positiveor negative change in circulating half-life of a modified insulinrelative to its non-modified form. Serum half-life is measured by takingblood samples at various time points after administration of insulin,and determining the concentration of that molecule in each sample.Correlation of the serum concentration with time allows calculation ofthe serum half-life. Increased serum half-life desirably has at leastabout two-fold, but a smaller increase may be useful, for example whereit enables a satisfactory dosing regimen or avoids a toxic effect. Insome embodiments, the increase is at least about three-fold, at leastabout five-fold, or at least about ten-fold.

The term “modulated therapeutic half-life” as used herein means thepositive or negative change in the half-life of the therapeuticallyeffective amount of insulin, relative to its non-modified form.Therapeutic half-life is measured by measuring pharmacokinetic and/orpharmacodynamic properties of the molecule at various time points afteradministration. Increased therapeutic half-life desirably enables aparticular beneficial dosing regimen, a particular beneficial totaldose, or avoids an undesired effect. In some embodiments, the increasedtherapeutic half-life results from increased potency, increased ordecreased binding of the modified molecule to its target, increased ordecreased breakdown of the molecule by enzymes such as proteases, or anincrease or decrease in another parameter or mechanism of action of thenon-modified molecule or an increase or decrease in receptor-mediatedclearance of the molecule.

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is free of at least some of thecellular components with which it is associated in the natural state, orthat the nucleic acid or protein has been concentrated to a levelgreater than the concentration of its in vivo or in vitro production. Itcan be in a homogeneous state. Isolated substances can be in either adry or semi-dry state, or in solution, including but not limited to, anaqueous solution. It can be a component of a pharmaceutical compositionthat comprises additional pharmaceutically acceptable carriers and/orexcipients. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinwhich is the predominant species present in a preparation issubstantially purified. In particular, an isolated gene is separatedfrom open reading frames which flank the gene and encode a protein otherthan the gene of interest. The term “purified” denotes that a nucleicacid or protein gives rise to substantially one band in anelectrophoretic gel. Particularly, it may mean that the nucleic acid orprotein is at least 85% pure, at least 90% pure, at least 95% pure, atleast 99% or greater pure.

The term “nucleic acid” refers to deoxyribonucleotides,deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymersthereof in either single- or double-stranded form. Unless specificallylimited, the term encompasses nucleic acids containing known analoguesof natural nucleotides which have similar binding properties as thereference nucleic acid and are metabolized in a manner similar tonaturally occurring nucleotides. Unless specifically limited otherwise,the term also refers to oligonucleotide analogs including PNA(peptidonucleic acid), analogs of DNA used in antisense technology(phosphorothioates, phosphoroamidates, and the like). Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (including but notlimited to, degenerate codon substitutions) and complementary sequencesas well as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batter et al., NucleicAcid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues.That is, a description directed to a polypeptide applies equally to adescription of a peptide and a description of a protein, and vice versa.The terms apply to naturally occurring amino acid polymers as well asamino acid polymers in which one or more amino acid residues is anon-naturally encoded amino acid. As used herein, the terms encompassamino acid chains of any length, including full length proteins, whereinthe amino acid residues are linked by covalent peptide bonds.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Reference to an amino acidincludes, for example, naturally occurring proteogenic L-amino acids;D-amino acids, chemically modified amino acids such as amino acidvariants and derivatives; naturally occurring non-proteogenic aminoacids such as β-alanine, ornithine, etc.; and chemically synthesizedcompounds having properties known in the art to be characteristic ofamino acids. Examples of non-naturally occurring amino acids include,but are not limited to, α-methyl amino acids (e.g., α-methyl alanine),D-amino acids, histidine-like amino acids (e.g., α-amino-histidine,β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine andα-methyl-histidine), amino acids having an extra methylene in the sidechain (“homo” amino acids), and amino acids in which a carboxylic acidfunctional group in the side chain is replaced with a sulfonic acidgroup (e.g., cysteic acid). The incorporation of non-natural aminoacids, including synthetic non-native amino acids, substituted aminoacids, or one or more D-amino acids into the proteins of the presentinvention may be advantageous in a number of different ways. D-aminoacid-containing peptides, etc., exhibit increased stability in vitro orin vivo compared to L-amino acid-containing counterparts. Thus, theconstruction of peptides, etc., incorporating D-amino acids can beparticularly useful when greater intracellular stability is desired orrequired. More specifically, D-peptides, etc., are resistant toendogenous peptidases and proteases, thereby providing improvedbioavailability of the molecule, and prolonged lifetimes in vivo whensuch properties are desirable. Additionally, D-peptides, etc., cannot beprocessed efficiently for major histocompatibility complex classII-restricted presentation to T helper cells, and are therefore, lesslikely to induce humoral immune responses in the whole organism.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skill inthe art will recognize that each codon in a nucleic acid (except AUG,which is ordinarily the only codon for methionine, and TGG, which isordinarily the only codon for tryptophan) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

As to amino acid sequences, one of ordinary skill in the art willrecognize that individual substitutions, deletions or additions to anucleic acid, peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the deletion of an amino acid, addition of anamino acid, or substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are known to those of ordinary skill in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

Conservative substitution tables providing functionally similar aminoacids are known to those of ordinary skill in the art. The followingeight groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” if they have a percentage of amino acidresidues or nucleotides that are the same (i.e., about 60% identity,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, orabout 95% identity over a specified region), when compared and alignedfor maximum correspondence over a comparison window, or designatedregion as measured using one of the following sequence comparisonalgorithms (or other algorithms available to persons of ordinary skillin the art) or by manual alignment and visual inspection. Thisdefinition also refers to the complement of a test sequence. Theidentity can exist over a region that is at least about 50 amino acidsor nucleotides in length, or over a region that is 75-100 amino acids ornucleotides in length, or, where not specified, across the entiresequence of a polynucleotide or polypeptide. A polynucleotide encoding apolypeptide of the present invention, including homologs from speciesother than human, may be obtained by a process comprising the steps ofscreening a library under stringent hybridization conditions with alabeled probe having a polynucleotide sequence of the invention or afragment thereof, and isolating full-length cDNA and genomic clonescontaining said polynucleotide sequence. Such hybridization techniquesare well known to the skilled artisan.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are known to those of ordinary skill in the art. Optimalalignment of sequences for comparison can be conducted, including butnot limited to, by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, PASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Informationavailable at the World Wide Web at ncbi.nlm.nih.gov. The BLAST algorithmparameters W, T, and X determine the sensitivity and speed of thealignment. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4 anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of10, M=5, N=−4, and a comparison of both strands. The BLAST algorithm istypically performed with the “low complexity” filter turned off.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, or less than about0.01, or less than about 0.001.

The phrase “selectively (or specifically) hybridizes to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent hybridization conditions when thatsequence is present in a complex mixture (including but not limited to,total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to hybridizationof sequences of DNA, RNA, PNA, or other nucleic acid mimics, orcombinations thereof under conditions of low ionic strength and hightemperature as is known in the art. Typically, under stringentconditions a probe will hybridize to its target subsequence in a complexmixture of nucleic acid (including but not limited to, total cellular orlibrary DNA or RNA) but does not hybridize to other sequences in thecomplex mixture. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Probes, “Overview of principles of hybridization and thestrategy of nucleic acid assays” (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(Tm) for the specific sequence at a defined ionic strength pH. The Tm isthe temperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at Tm, 50% of the probes are occupied atequilibrium). Stringent conditions may be those in which the saltconcentration is less than about 1.0 M sodium ion., typically about 0.01to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 andthe temperature is at least about 30° C. for short probes (including butnot limited to, 10 to 50 nucleotides) and at least about 60° C. for longprobes (including but not limited to, greater than 50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. For selective or specifichybridization, a positive signal may be at least two times background,optionally 10 times background hybridization. Exemplary stringenthybridization conditions can be as following: 50% formamide, 5×SSC, and1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C.,with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can beperformed for 5, 15, 30, 60, 120, or more minutes.

As used herein, the term “eukaryote” refers to organisms belonging tothe phylogenetic domain Eucarya such as animals (including but notlimited to, mammals, insects, reptiles, birds, etc.), ciliates, plants(including but not limited to, monocots, dicots, algae, etc.), fungi,yeasts, flagellates, microsporidia, protists, etc.

As used herein, the term “non-eukaryote” refers to non-eukaryoticorganisms. For example, a non-eukaryotic organism can belong to theEubacteria (including but not limited to, Escherichia coli, Thermusthermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens,Pseudomonas aeruginosa, Pseudomonas putida, etc.) phylogenetic domain,or the Archaea (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium such as Haloferaxvolcanii and Halobacterium species NRC-1, Arehaeoglobus fulgidus,Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.)phylogenetic domain.

The term “subject” as used herein, refers to an animal, in someembodiments a mammal, and in other embodiments a human, who is theobject of treatment, observation or experiment. An animal may be acompanion animal (e.g., dogs, cats, and the like), farm animal (e.g.,cows, sheep, pigs, horses, and the like) or a laboratory animal (e.g.,rats, mice, guinea pigs, and the like).

The term “effective amount” as used herein refers to that amount of themodified non-natural amino acid polypeptide being administered whichwill relieve to some extent one or more of the symptoms of the disease,condition or disorder being treated. Compositions containing themodified non-natural amino acid polypeptide described herein can beadministered for prophylactic, enhancing, and/or therapeutic treatments.

The terms “enhance” or “enhancing” means to increase or prolong eitherin potency or duration a desired effect. Thus, in regard to enhancingthe effect of therapeutic agents, the term “enhancing” refers to theability to increase or prolong, either in potency or duration, theeffect of other therapeutic agents on a system. An “enhancing-effectiveamount,” as used herein, refers to an amount adequate to enhance theeffect of another therapeutic agent in a desired system. When used in apatient, amounts effective for this use will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician.

The term “modified,” as used herein refers to any changes made to agiven polypeptide, such as changes to the length of the polypeptide, theamino acid sequence, chemical structure, co-translational modification,or post-translational modification of a polypeptide. The form“(modified)” term means that the polypeptides being discussed areoptionally modified, that is, the polypeptides under discussion can bemodified or unmodified.

The term “post-translationally modified” refers to any modification of anatural or non-natural amino acid that occurs to such an amino acidafter it has been incorporated into a polypeptide chain. The termencompasses, by way of example only, co-translational in vivomodifications, co-translational in vitro modifications (such as in acell-free translation system), post-translational in vivo modifications,and post-translational in vitro modifications.

In prophylactic applications, compositions containing the insulinpolypeptide are administered to a patient susceptible to or otherwise atrisk of a particular disease, disorder or condition. Such an amount isdefined to be a “prophylactically effective amount.” In this use, theprecise amounts also depend on the patient's state of health, weight,and the like. It is considered well within the skill of the art for oneto determine such prophylactically effective amounts by routineexperimentation (e.g., a dose escalation clinical trial).

The term “protected” refers to the presence of a “protecting group” ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin or with the methods and compositions described herein, includingphotolabile groups such as Nvoc and MeNvoc. Other protecting groupsknown in the art may also be used in or with the methods andcompositions described herein.

By way of example only, blocking/protecting groups may be selected from:

Other protecting groups are described in Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, N.Y.,1999, which is incorporated herein by reference in its entirety.

In therapeutic applications, compositions containing the modifiednon-natural amino acid polypeptide are administered to a patient alreadysuffering from a disease, condition or disorder, in an amount sufficientto cure or at least partially arrest the symptoms of the disease,disorder or condition. Such an amount is defined to be a“therapeutically effective amount,” and will depend on the severity andcourse of the disease, disorder or condition, previous therapy, thepatient's health status and response to the drugs, and the judgment ofthe treating physician. It is considered well within the skill of theart for one to determine such therapeutically effective amounts byroutine experimentation (e.g., a dose escalation clinical trial).

The term “treating” is used to refer to either prophylactic and/ortherapeutic treatments.

Non-naturally encoded amino acid polypeptides presented herein mayinclude isotopically-labelled compounds with one or more atoms replacedby an atom having an atomic mass or mass number different from theatomic mass or mass number usually found in nature. Examples of isotopesthat can be incorporated into the present compounds include isotopes ofhydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as 2H,3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl, respectively. Certainisotopically-labelled compounds described herein, for example those intowhich radioactive isotopes such as 3H and 14C are incorporated, may beuseful in drug and/or substrate tissue distribution assays. Further,substitution with isotopes such as deuterium, i.e., 2H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements.

All isomers including but not limited to diastereomers, enantiomers, andmixtures thereof are considered as part of the compositions describedherein. In additional or further embodiments, the non-naturally encodedamino acid polypeptides are metabolized upon administration to anorganism in need to produce a metabolite that is then used to produce adesired effect, including a desired therapeutic effect. In further oradditional embodiments are active metabolites of non-naturally encodedamino acid polypeptides.

In some situations, non-naturally encoded amino acid polypeptides mayexist as tautomers. In addition, the non-naturally encoded amino acidpolypeptides described herein can exist in unsolvated as well assolvated forms with pharmaceutically acceptable solvents such as water,ethanol, and the like. The solvated forms are also considered to bedisclosed herein. Those of ordinary skill in the art will recognize thatsome of the compounds herein can exist in several tautomeric forms. Allsuch tautomeric forms are considered as part of the compositionsdescribed herein.

Unless otherwise indicated, conventional methods of mass spectroscopy,NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniquesand pharmacology, within the skill of the art are employed.

DETAILED DESCRIPTION I. Introduction

Insulin polypeptides comprising at least one unnatural amino acid areprovided in the invention. In certain embodiments of the invention, theinsulin polypeptide with at least one unnatural amino acid includes atleast one post-translational modification. In one embodiment, the atleast one post-translational modification comprises attachment of amolecule including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide, aradiotransmitter, a neutron-capture agent, or any combination of theabove or any other desirable compound or substance, comprising a secondreactive group to at least one unnatural amino acid comprising a firstreactive group utilizing chemistry methodology that is known to one ofordinary skill in the art to be suitable for the particular reactivegroups. For example, the first reactive group is an alkynyl moiety(including but not limited to, in the unnatural amino acidp-propargyloxyphenylalanine, where the propargyl group is also sometimesreferred to as an acetylene moiety) and the second reactive group is anazido moiety, and [3+2] cycloaddition chemistry methodologies areutilized. In another example, the first reactive group is the azidomoiety (including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety. In certain embodiments of the modified insulin polypeptide ofthe present invention, at least one unnatural amino acid (including butnot limited to, unnatural amino acid containing a keto functional group)comprising at least one post-translational modification, is used wherethe at least one post-translational modification comprises a saccharidemoiety. In certain embodiments, the post-translational modification ismade in vivo in a eukaryotic cell or in a non-eukaryotic cell. A linker,polymer, water soluble polymer, or other molecule may attach themolecule to the polypeptide. The molecule may be linked directly to thepolypeptide.

In certain embodiments, the protein includes at least onepost-translational modification that is made in vivo by one host cell,where the post-translational modification is not normally made byanother host cell type. In certain embodiments, the protein includes atleast one post-translational modification that is made in vivo by aeukaryotic cell, where the post-translational modification is notnormally made by a non-eukaryotic cell. Examples of post-translationalmodifications include, but are not limited to, glycosylation,acetylation, acylation, lipid-modification, palmitoylation, palmitateaddition, phosphorylation, glycolipid-linkage modification, and thelike.

In some embodiments, the insulin polypeptide comprises one or morenon-naturally encoded amino acids for glycosylation, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, or glycolipid-linkage modification of the polypeptide.In some embodiments, the insulin polypeptide comprises one or morenon-naturally encoded amino acids for glycosylation of the polypeptide.In some embodiments, the insulin polypeptide comprises one or morenaturally encoded amino acids for glycosylation, acetylation, acylation,lipid-modification, palmitoylation, palmitate addition, phosphorylation,or glycolipid-linkage modification of the polypeptide. In someembodiments, the insulin polypeptide comprises one or more naturallyencoded amino acids for glycosylation of the polypeptide.

In some embodiments, the insulin polypeptide comprises one or morenon-naturally encoded amino acid additions and/or substitutions thatenhance glycosylation of the polypeptide. In some embodiments, theinsulin polypeptide comprises one or more deletions that enhanceglycosylation of the polypeptide. In some embodiments, the insulinpolypeptide comprises one or more non-naturally encoded amino acidadditions and/or substitutions that enhance glycosylation at a differentamino acid in the polypeptide. In some embodiments, the insulinpolypeptide comprises one or more deletions that enhance glycosylationat a different amino acid in the polypeptide. In some embodiments, theinsulin polypeptide comprises one or more non-naturally encoded aminoacid additions and/or substitutions that enhance glycosylation at anon-naturally encoded amino acid in the polypeptide. In someembodiments, the insulin polypeptide comprises one or more non-naturallyencoded amino acid additions and/or substitutions that enhanceglycosylation at a naturally encoded amino acid in the polypeptide. Insome embodiments, the insulin polypeptide comprises one or morenaturally encoded amino acid additions and/or substitutions that enhanceglycosylation at a different amino acid in the polypeptide. In someembodiments, the insulin polypeptide comprises one or more non-naturallyencoded amino acid additions and/or substitutions that enhanceglycosylation at a naturally encoded amino acid in the polypeptide. Insome embodiments, the insulin polypeptide comprises one or morenon-naturally encoded amino acid additions and/or substitutions thatenhance glycosylation at a non-naturally encoded amino acid in thepolypeptide.

In one embodiment, the post-translational modification comprisesattachment of an oligosaccharide to an asparagine by a GlcNAc-asparaginelinkage (including but not limited to, where the oligosaccharidecomprises (GlcNAc-Man)2-Man-GlcNAc-GlcNAc, and the like). In anotherembodiment, the post-translational modification comprises attachment ofan oligosaccharide (including but not limited to, Gal-GalNAc,Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc-serine, aGalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. Incertain embodiments, a protein or polypeptide of the invention cancomprise a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, and/or the like. Examples ofsecretion signal sequences include, but are not limited to, aprokaryotic secretion signal sequence, a eukaryotic secretion signalsequence, a eukaryotic secretion signal sequence 5′-optimized forbacterial expression, a novel secretion signal sequence, pectate lyasesecretion signal sequence, Omp A secretion signal sequence, and a phagesecretion signal sequence. Examples of secretion signal sequences,include, but are not limited to, STII (prokaryotic), Fd GIII and M13(phage), Bgl2 (yeast), and the signal sequence bla derived from atransposon. Any such sequence may be modified to provide a desiredresult with the polypeptide, including but not limited to, substitutingone signal sequence with a different signal sequence, substituting aleader sequence with a different leader sequence, etc.

The protein or polypeptide of interest can contain at least one, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or ten or more unnaturalamino acids. The unnatural amino acids can be the same or different, forexample, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more differentsites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moredifferent unnatural amino acids. In certain embodiments, at least one,but fewer than all, of a particular amino acid present in a naturallyoccurring version of the protein is substituted with an unnatural aminoacid.

The present invention provides methods and compositions based on insulincomprising at least one non-naturally encoded amino acid. Introductionof at least one non-naturally encoded amino acid into insulin can allowfor the application of conjugation chemistries that involve specificchemical reactions, including, but not limited to, with one or morenon-naturally encoded amino acids while not reacting with the commonlyoccurring 20 amino acids. In some embodiments, insulin comprising thenon-naturally encoded amino acid is linked to a water soluble polymer,such as polyethylene glycol (PEG), via the side chain of thenon-naturally encoded amino acid. This invention provides a highlyefficient method for the selective modification of proteins with PEGderivatives, which involves the selective incorporation ofnon-genetically encoded amino acids, including but not limited to, thoseamino acids containing functional groups or substituents not found inthe 20 naturally incorporated amino acids, including but not limited toa ketone, an azide or acetylene moiety, into proteins in response to aselector codon and the subsequent modification of those amino acids witha suitably reactive PEG derivative. Once incorporated, the amino acidside chains can then be modified by utilizing chemistry methodologiesknown to those of ordinary skill in the art to be suitable for theparticular functional groups or substituents present in thenon-naturally encoded amino acid. Known chemistry methodologies of awide variety are suitable for use in the present invention toincorporate a water soluble polymer into the protein. Such methodologiesinclude but are not limited to a Huisgen [3+2] cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, acetylene or azidederivatives, respectively.

Because the Huisgen [3+2] cycloaddition method involves a cycloadditionrather than a nucleophilic substitution reaction, proteins can bemodified with extremely high selectivity. The reaction can be carriedout at room temperature in aqueous conditions with excellentregioselectivity (1,4>1,5) by the addition of catalytic amounts of Cu(I)salts to the reaction mixture. See, e.g., Tornoe, et al., (2002) J. Org.Chem. 67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int.Ed. 41:2596-2599; and WO 03/101972. A molecule that can be added to aprotein of the invention through a [3+2] cycloaddition includesvirtually any molecule with a suitable functional group or substituentincluding but not limited to an azido or acetylene derivative. Thesemolecules can be added to an unnatural amino acid with an acetylenegroup, including but not limited to, p-propargyloxyphenylalanine, orazido group, including but not limited to p-azido-phenylalanine,respectively.

The five-membered ring that results from the Huisgen [3+2] cycloadditionis not generally reversible in reducing environments and is stableagainst hydrolysis for extended periods in aqueous environments.Consequently, the physical and chemical characteristics of a widevariety of substances can be modified under demanding aqueous conditionswith the active PEG derivatives of the present invention. Even moreimportantly, because the azide and acetylene moieties are specific forone another (and do not, for example, react with any of the 20 common,genetically-encoded amino acids), proteins can be modified in one ormore specific sites with extremely high selectivity.

The invention also provides water soluble and hydrolytically stablederivatives of PEG derivatives and related hydrophilic polymers havingone or more acetylene or azide moieties. The PEG polymer derivativesthat contain acetylene moieties are highly selective for coupling withazide moieties that have been introduced selectively into proteins inresponse to a selector codon. Similarly, PEG polymer derivatives thatcontain azide moieties are highly selective for coupling with acetylenemoieties that have been introduced selectively into proteins in responseto a selector codon.

More specifically, the azide moieties comprise, but are not limited to,alkyl azides, aryl azides and derivatives of these azides. Thederivatives of the alkyl and aryl azides can include other substituentsso long as the acetylene-specific reactivity is maintained. Theacetylene moieties comprise alkyl and aryl acetylenes and derivatives ofeach. The derivatives of the alkyl and aryl acetylenes can include othersubstituents so long as the azide-specific reactivity is maintained.

The present invention provides conjugates of substances having a widevariety of functional groups, substituents or moieties, with othersubstances including but not limited to a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a radionuclide; a cytotoxic compound; a drug; anaffinity label; a photoaffinity label; a reactive compound; a resin; asecond protein or polypeptide or polypeptide analog; an antibody orantibody fragment; a metal chelator; a cofactor; a fatty acid; acarbohydrate; a polynucleotide; a DNA; a RNA; an antisensepolynucleotide; a saccharide; a water-soluble dendrimer; a cyclodextrin;an inhibitory ribonucleic acid; a biomaterial; a nanoparticle; a spinlabel; a fluorophore, a metal-containing moiety; a radioactive moiety; anovel functional group; a group that covalently or noncovalentlyinteracts with other molecules; a photocaged moiety; an actinicradiation excitable moiety; a photoisomerizable moiety; biotin; aderivative of biotin; a biotin analogue; a moiety incorporating a heavyatom; a chemically cleavable group; a photocleavable group; an elongatedside chain; a carbon-linked sugar; a redox-active agent; an aminothioacid; a toxic moiety; an isotopically labeled moiety; a biophysicalprobe; a phosphorescent group; a chemiluminescent group; an electrondense group; a magnetic group; an intercalating group; a chromophore; anenergy transfer agent; a biologically active agent; a detectable label;a small molecule; a quantum dot; a nanotransmitter; a radionucleotide; aradiotransmitter; a neutron-capture agent; or any combination of theabove, or any other desirable compound or substance. The presentinvention also includes conjugates of substances having azide oracetylene moieties with PEG polymer derivatives having the correspondingacetylene or azide moieties. For example, a PEG polymer containing anazide moiety can be coupled to a biologically active molecule at aposition in the protein that contains a non-genetically encoded aminoacid bearing an acetylene functionality. The linkage by which the PEGand the biologically active molecule are coupled includes but is notlimited to the Huisgen [3+2] cycloaddition product.

It is well established in the art that PEG can be used to modify thesurfaces of biomaterials (see, e.g., U.S. Pat. No. 6,610,281; Mehvar,R., J. Pharm Pharm Sci., 3(1):125-136 (2000) which are incorporated byreference herein). The invention also includes biomaterials comprising asurface having one or more reactive azide or acetylene sites and one ormore of the azide- or acetylene-containing polymers of the inventioncoupled to the surface via the Huisgen [3+2] cycloaddition linkage.Biomaterials and other substances can also be coupled to the azide- oracetylene-activated polymer derivatives through a linkage other than theazide or acetylene linkage, such as through a linkage comprising acarboxylic acid, amine, alcohol or thiol moiety, to leave the azide oracetylene moiety available for subsequent reactions.

The invention includes a method of synthesizing the azide- andacetylene-containing polymers of the invention. In the case of theazide-containing PEG derivative, the azide can be bonded directly to acarbon atom of the polymer. Alternatively, the azide-containing PEGderivative can be prepared by attaching a linking agent that has theazide moiety at one terminus to a conventional activated polymer so thatthe resulting polymer has the azide moiety at its terminus. In the caseof the acetylene-containing PEG derivative, the acetylene can be bondeddirectly to a carbon atom of the polymer. Alternatively, theacetylene-containing PEG derivative can be prepared by attaching alinking agent that has the acetylene moiety at one terminus to aconventional activated polymer so that the resulting polymer has theacetylene moiety at its terminus.

More specifically, in the case of the azide-containing PEG derivative, awater soluble polymer having at least one active hydroxyl moietyundergoes a reaction to produce a substituted polymer having a morereactive moiety, such as a mesylate, tresylate, tosylate or halogenleaving group, thereon. The preparation and use of PEG derivativescontaining sulfonyl acid halides, halogen atoms and other leaving groupsare known to those of ordinary skill in the art. The resultingsubstituted polymer then undergoes a reaction to substitute for the morereactive moiety an azide moiety at the terminus of the polymer.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an azide at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the azidemoiety is positioned at the terminus of the polymer. Nucleophilic andelectrophilic moieties, including amines, thiols, hydrazides,hydrazines, alcohols, carboxylates, aldehydes, ketones, thioesters andthe like, are known to those of ordinary skill in the art.

More specifically, in the case of the acetylene-containing PEGderivative, a water soluble polymer having at least one active hydroxylmoiety undergoes a reaction to displace a halogen or other activatedleaving group from a precursor that contains an acetylene moiety.Alternatively, a water soluble polymer having at least one activenucleophilic or electrophilic moiety undergoes a reaction with a linkingagent that has an acetylene at one terminus so that a covalent bond isformed between the PEG polymer and the linking agent and the acetylenemoiety is positioned at the terminus of the polymer. The use of halogenmoieties, activated leaving group, nucleophilic and electrophilicmoieties in the context of organic synthesis and the preparation and useof PEG derivatives is well established to practitioners in the art.

The invention also provides a method for the selective modification ofproteins to add other substances to the modified protein, including butnot limited to water soluble polymers such as PEG and PEG derivativescontaining an azide or acetylene moiety. The azide- andacetylene-containing PEG derivatives can be used to modify theproperties of surfaces and molecules where biocompatibility, stability,solubility and lack of immunogenicity are important, while at the sametime providing a more selective means of attaching the PEG derivativesto proteins than was previously known in the art.

General Recombinant Nucleic Acid Methods for Use with the Invention

In numerous embodiments of the present invention, nucleic acids encodingan insulin polypeptide of interest will be isolated, cloned and oftenaltered using recombinant methods. Such embodiments are used, includingbut not limited to, for protein expression or during the generation ofvariants, derivatives, expression cassettes, or other sequences derivedfrom an insulin polypeptide. In some embodiments, the sequences encodingthe polypeptides of the invention are operably linked to a heterologouspromoter.

A nucleotide sequence encoding an insulin polypeptide comprising anon-naturally encoded amino acid may be synthesized on the basis of theamino acid sequence of the parent polypeptide, including but not limitedto, having the amino acid sequence shown in SEQ ID NO: 1 and SEQ ID NO:2 and then changing the nucleotide sequence so as to effect introduction(i.e., incorporation or substitution) or removal (i.e., deletion orsubstitution) of the relevant amino acid residue(s). The nucleotidesequence may be conveniently modified by site-directed mutagenesis inaccordance with conventional methods. Alternatively, the nucleotidesequence may be prepared by chemical synthesis, including but notlimited to, by using an oligonucleotide synthesizer, whereinoligonucleotides are designed based on the amino acid sequence of thedesired polypeptide, and preferably selecting those codons that arefavored in the host cell in which the recombinant polypeptide will beproduced.

This invention utilizes routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, includingbut not limited to, the generation of genes or polynucleotides thatinclude selector codons for production of proteins that includeunnatural amino acids, orthogonal tRNAs, orthogonal synthetases, andpairs thereof.

Various types of mutagenesis are used in the invention for a variety ofpurposes, including but not limited to, to produce novel synthetases ortRNAs, to mutate tRNA molecules, to mutate polynucleotides encodingsynthetases, to produce libraries of tRNAs, to produce libraries ofsynthetases, to produce selector codons, to insert selector codons thatencode unnatural amino acids in a protein or polypeptide of interest.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination, DNA shuffling or other recursivemutagenesis methods, chimeric construction, mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like, PCT-mediated mutagenesis, or any combinationthereof. Additional suitable methods include point mismatch repair,mutagenesis using repair-deficient host strains, restriction-selectionand restriction-purification, deletion mutagenesis, mutagenesis by totalgene synthesis, double-strand break repair, and the like. Mutagenesis,including but not limited to, involving chimeric constructs, are alsoincluded in the present invention. In one embodiment, mutagenesis can beguided by known information of the naturally occurring molecule oraltered or mutated naturally occurring molecule, including but notlimited to, sequence, sequence comparisons, physical properties,secondary, tertiary, or quaternary structure, crystal structure or thelike.

The texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Zoller & Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors: an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller & Smith, Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods inEnzymol. 100:468-500 (1983); Zoller & Smith, Oligonucleotide-directedmutagenesis: a simple method using two oligonucleotide primers and asingle-stranded DNA template, Methods in Enzymol. 154:329-350 (1987);Taylor et al., The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA, Nucl. Acids Res. 13: 8749-8764(1985); Taylor et al., The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstein, Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 14: 9679-9698 (1986); Sayers et al., 5′-3′ Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl. AcidsRes. 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Different base/base mismatches arecorrected with different efficiencies by the methyl-directed DNAmismatch-repair system of E. coli, Cell 38:879-887 (1984); Carter etal., Improved oligonucleotide site-directed mutagenesis using M13vectors, Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using M13 vectors, Methods inEnzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use ofoligonucleotides to generate large deletions, Nucl. Acids Res. 14: 5115(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakmar and Khorana, Total synthesis and expression of a gene forthe alpha-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001); W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of synthetases, or altering tRNAs, aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage and Caruthers,Tetrahedron Letts. 22(20):1859-1862, (1981) e.g., using an automatedsynthesizer, as described in Needham-VanDevanter et al, Nucleic AcidsRes., 12:6159-6168 (1984).

The invention also relates to eukaryotic host cells, non-eukaryotic hostcells, and organisms for the in vivo incorporation of an unnatural aminoacid via orthogonal tRNA/RS pairs. Host cells are genetically engineered(including but not limited to, transformed, transduced or transfected)with the polynucleotides of the invention or constructs which include apolynucleotide of the invention, including but not limited to, a vectorof the invention, which can be, for example, a cloning vector or anexpression vector. For example, the coding regions for the orthogonaltRNA, the orthogonal tRNA synthetase, and the protein to be derivatizedare operably linked to gene expression control elements that arefunctional in the desired host cell. The vector can be, for example, inthe form of a plasmid, a cosmid, a phage, a bacterium, a virus, a nakedpolynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)), and/or the like. Techniques suitable for thetransfer of nucleic acid into cells in vitro include the use ofliposomes, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. In vivo gene transfer techniquesinclude, but are not limited to, transfection with viral (typicallyretroviral) vectors and viral coat protein-liposome mediatedtransfection [Dzau et al., Trends in Biotechnology 11:205-210 (1993)].In some situations it may be desirable to provide the nucleic acidsource with an agent that targets the target cells, such as an antibodyspecific for a cell surface membrane protein or the target cell, aligand for a receptor on the target cell, etc. Where liposomes areemployed, proteins which bind to a cell surface membrane proteinassociated with endocytosis may be used for targeting and/or tofacilitate uptake, e.g. capsid proteins or fragments thereof tropic fora particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms. Other usefulreferences, including but not limited to for cell isolation and culture(e.g., for subsequent nucleic acid isolation) include Freshney (1994)Culture of Animal Cells, a Manual of Basic Technique, third edition,Wiley-Liss, New York and the references cited therein; Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

Several well-known methods of introducing target nucleic acids intocells are available, any of which can be used in the invention. Theseinclude: fusion of the recipient cells with bacterial protoplastscontaining the DNA, electroporation, projectile bombardment, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™ from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (including but not limited to,shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or both. See, Gillam & Smith, Gene 8:81(1979); Roberts, et al., Nature, 328:731 (1987); Schneider, E., et al.,Protein Expr. Purif. 6(1):10-14 (1995); Ausubel, Sambrook, Berger (allsupra). A catalogue of bacteria and bacteriophages useful for cloning isprovided, e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Ghema et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY. In addition, essentially any nucleic acid (andvirtually any labeled nucleic acid, whether standard or non-standard)can be custom or standard ordered from any of a variety of commercialsources, such as the Midland Certified Reagent Company (Midland, Tex.available on the World Wide Web at mcrc.com), The Great American GeneCompany (Ramona, Calif. available on the World Wide Web at genco.com),ExpressGen Inc. (Chicago, Ill. available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, Calif.) and manyothers.

Selector Codons

Selector codons of the invention expand the genetic codon framework ofprotein biosynthetic machinery. For example, a selector codon includes,but is not limited to, a unique three base codon, a nonsense codon, suchas a stop codon, including but not limited to, an amber codon (UAG), anochre codon, or an opal codon (UGA), an unnatural codon, a four or morebase codon, a rare codon, or the like. It is readily apparent to thoseof ordinary skill in the art that there is a wide range in the number ofselector codons that can be introduced into a desired gene orpolynucleotide, including but not limited to, one or more, two or more,three or more, 4, 5, 6, 7, 8, 9, 10 or more in a single polynucleotideencoding at least a portion of the insulin polypeptide. It is alsoreadily apparent to those of ordinary skill in the art that there is awide range in the number of selector codons that can be introduced intoa desired gene or polynucleotide, including but not limited to, one ormore, two or more, three or more, 4, 5, 6, 7, 8, 9, 10 or more totalfound in the A chain and B chain polynucleotide sequences encoding atleast a portion of the insulin polypeptide.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of one or more unnatural aminoacids in vivo. For example, an O-tRNA is produced that recognizes thestop codon, including but not limited to, UAG, and is aminoacylated byan O-RS with a desired unnatural amino acid. This O-tRNA is notrecognized by the naturally occurring host's aminoacyl-tRNA synthetases.Conventional site-directed mutagenesis can be used to introduce the stopcodon, including but not limited to, TAG, at the site of interest in apolypeptide of interest. See, e.g., Sayers, J. R., et al. (1988), 5′-3′Exonucleases in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 16:791-802. When the O-RS, O-tRNA andthe nucleic acid that encodes the polypeptide of interest are combinedin vivo, the unnatural amino acid is incorporated in response to the UAGcodon to give a polypeptide containing the unnatural amino acid at thespecified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the eukaryotic host cell. For example,because the suppression efficiency for the UAG codon depends upon thecompetition between the O-tRNA, including but not limited to, the ambersuppressor tRNA, and a eukaryotic release factor (including but notlimited to, eRF) (which binds to a stop codon and initiates release ofthe growing peptide from the ribosome), the suppression efficiency canbe modulated by, including but not limited to, increasing the expressionlevel of O-tRNA, and/or the suppressor tRNA.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., Ma et al., Biochemistry, 32:7939 (1993). In thiscase, the synthetic tRNA competes with the naturally occurring tRNAArg,which exists as a minor species in Escherichia coli. Some organisms donot use all triplet codons. An unassigned codon AGA in Micrococcusluteus has been utilized for insertion of amino acids in an in vitrotranscription/translation extract. See, e.g., Kowal and Oliver, Nucl.Acid. Res., 25:4685 (1997). Components of the present invention can begenerated to use these rare codons in vivo.

Selector codons also comprise extended codons, including but not limitedto, four or more base codons, such as, four, five, six or more basecodons. Examples of four base codons include, but are not limited to,AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU,UAGGC and the like. A feature of the invention includes using extendedcodons based on frameshift suppression. Four or more base codons caninsert, including but not limited to, one or multiple unnatural aminoacids into the same protein. For example, in the presence of mutatedO-tRNAs, including but not limited to, a special frameshift suppressortRNAs, with anticodon loops, for example, with at least 8-10 ntanticodon loops, the four or more base codon is read as single aminoacid. In other embodiments, the anticodon loops can decode, includingbut not limited to, at least a four-base codon, at least a five-basecodon, or at least a six-base codon or more. Since there are 256possible four-base codons, multiple unnatural amino acids can be encodedin the same cell using a four or more base codon. See, Anderson et al.,(2002) Exploring the Limits of Codon and Anticodon Size, Chemistry andBiology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769.

For example, four-base codons have been used to incorporate unnaturalamino acids into proteins using in vitro biosynthetic methods. See,e.g., Ma et al., (1993) Biochemistry, 32:7939; and Hohsaka et al.,(1999) J. Am. Chem. Soc., 121:34. CGGG and AGGU were used tosimultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., Hohsaka et al., (1999) J. Am.Chem. Soc., 121:12194. In an in vivo study, Moore et al. examined theability of tRNALeu derivatives with NCUA anticodons to suppress UAGNcodons (N can be U, A, G, or C), and found that the quadruplet UAGA canbe decoded by a tRNALeu with a UCUA anticodon with an efficiency of 13to 26% with little decoding in the 0 or −1 frame. See, Moore et al.,(2000) J. Mol. Biol., 298:195. In one embodiment, extended codons basedon rare codons or nonsense codons can be used in the present invention,which can reduce missense readthrough and frameshift suppression atother unwanted sites.

For a given system, a selector codon can also include one of the naturalthree base codons, where the endogenous system does not use (or rarelyuses) the natural base codon. For example, this includes a system thatis lacking a tRNA that recognizes the natural three base codon, and/or asystem where the three base codon is a rare codon.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Him, et al., (2002)An unnatural base pair for incorporating amino acid analogues intoprotein, Nature Biotechnology, 20:177-182. See, also, Wu, Y., et al.,(2002) J. Am. Chem. Soc. 124:14626-14630. Other relevant publicationsare listed below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., Switzer et al., (1989) J. Am. Chem. Soc.,111:8322; and Piccirilli et al., (1990) Nature, 343:33; Kool, (2000)Curr. Opin. Chem. Biol., 4:602. These bases in general mispair to somedegree with natural bases and cannot be enzymatically replicated. Kooland co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, Kool, (2000) Curr. Opin. Chem. Biol., 4:602; and Guckianand Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort todevelop an unnatural base pair satisfying all the above requirements,Schultz, Romesberg and co-workers have systematically synthesized andstudied a series of unnatural hydrophobic bases. A PICS:PICS self-pairis found to be more stable than natural base pairs, and can beefficiently incorporated into DNA by Klenow fragment of Escherichia coliDNA polymerase I (KF). See, e.g., McMinn et al., (1999) J. Am. Chem.Soc., 121:11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc., 122:3274.A 3MN:3MN self-pair can be synthesized by KF with efficiency andselectivity sufficient for biological function. See, e.g., Ogawa et al.,(2000) J. Am. Chem. Soc., 122:8803. However, both bases act as a chainterminator for further replication. A mutant DNA polymerase has beenrecently evolved that can be used to replicate the PICS self pair. Inaddition, a 7AI self pair can be replicated. See, e.g., The et al.,(2001) J. Am. Chem. Soc., 123:7439. A novel metallobase pair, Dipic:Py,has also been developed, which forms a stable pair upon binding Cu(II).See, Meggers et al., (2000) J. Am. Chem. Soc., 122:10714. Becauseextended codons and unnatural codons are intrinsically orthogonal tonatural codons, the methods of the invention can take advantage of thisproperty to generate orthogonal tRNAs for them.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is incorporated into a gene but isnot translated into protein. The sequence contains a structure thatserves as a cue to induce the ribosome to hop over the sequence andresume translation downstream of the insertion.

In certain embodiments, the protein or polypeptide of interest (orportion thereof) in the methods and/or compositions of the invention isencoded by a nucleic acid. Typically, the nucleic acid comprises atleast one selector codon, at least two selector codons, at least threeselector codons, at least four selector codons, at least five selectorcodons, at least six selector codons, at least seven selector codons, atleast eight selector codons, at least nine selector codons, ten or moreselector codons.

Genes coding for proteins or polypeptides of interest can be mutagenizedusing methods known to one of ordinary skill in the art and describedherein to include, for example, one or more selector codon for theincorporation of an unnatural amino acid. For example, a nucleic acidfor a protein of interest is mutagenized to include one or more selectorcodon, providing for the incorporation of one or more unnatural aminoacids. The invention includes any such variant, including but notlimited to, mutant, versions of any protein, for example, including atleast one unnatural amino acid. Similarly, the invention also includescorresponding nucleic acids, i.e., any nucleic acid with one or moreselector codon that encodes one or more unnatural amino acid.

Nucleic acid molecules encoding a protein of interest such as an insulinpolypeptide may be readily mutated to introduce a cysteine at anydesired position of the polypeptide. Cysteine is widely used tointroduce reactive molecules, water soluble polymers, proteins, or awide variety of other molecules, onto a protein of interest. Methodssuitable for the incorporation of cysteine into a desired position of apolypeptide are known to those of ordinary skill in the art, such asthose described in U.S. Pat. No. 6,608,183, which is incorporated byreference herein, and standard mutagenesis techniques.

Non-Naturally Encoded Amino Acids

A very wide variety of non-naturally encoded amino acids are suitablefor use in the present invention. Any number of non-naturally encodedamino acids can be introduced into an insulin polypeptide. In general,the introduced non-naturally encoded amino acids are substantiallychemically inert toward the 20 common, genetically-encoded amino acids(i.e., alanine, arginine, asparagine, aspartic acid, cysteine,glutamine, glutamic acid, glycine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine). In some embodiments, thenon-naturally encoded amino acids include side chain functional groupsthat react efficiently and selectively with functional groups not foundin the 20 common amino acids (including but not limited to, azido,ketone, aldehyde and aminooxy groups) to form stable conjugates. Forexample, an insulin polypeptide that includes a non-naturally encodedamino acid containing an azido functional group can be reacted with apolymer (including but not limited to, polyethylene glycol) or,alternatively, a second polypeptide containing an alkyne moiety to forma stable conjugate resulting for the selective reaction of the azide andthe alkyne functional groups to form a Huisgen [3+2] cycloadditionproduct.

The generic structure of an alpha-amino acid is illustrated as follows(Formula I):

A non-naturally encoded amino acid is typically any structure having theabove-listed formula wherein the R group is any substituent other thanone used in the twenty natural amino acids, and may be suitable for usein the present invention. Because the non-naturally encoded amino acidsof the invention typically differ from the natural amino acids only inthe structure of the side chain, the non-naturally encoded amino acidsform amide bonds with other amino acids, including but not limited to,natural or non-naturally encoded, in the same manner in which they areformed in naturally occurring polypeptides.

However, the non-naturally encoded amino acids have side chain groupsthat distinguish them from the natural amino acids. For example, Roptionally comprises an alkyl-, aryl-, acyl-, keto-, azido-, hydroxyl-,hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether, thiol,seleno-, sulfonyl-, borate, boronate, phospho, phosphono, phosphine,heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine,amino group, or the like or any combination thereof. Other non-naturallyoccurring amino acids of interest that may be suitable for use in thepresent invention include, but are not limited to, amino acidscomprising a photoactivatable cross-linker, spin-labeled amino acids,fluorescent amino acids, metal binding amino acids, metal-containingamino acids, radioactive amino acids, amino acids with novel functionalgroups, amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, amino acidscomprising biotin or a biotin analogue, glycosylated amino acids such asa sugar substituted serine, other carbohydrate modified amino acids,keto-containing amino acids, amino acids comprising polyethylene glycolor polyether, heavy atom substituted amino acids, chemically cleavableand/or photocleavable amino acids, amino acids with an elongated sidechains as compared to natural amino acids, including but not limited to,polyethers or long chain hydrocarbons, including but not limited to,greater than about 5 or greater than about 10 carbons, carbon-linkedsugar-containing amino acids, redox-active amino acids, amino thioacidcontaining amino acids, and amino acids comprising one or more toxicmoiety.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of ordinary skill in the art. Fororganic synthesis techniques, see, e.g., Organic Chemistry by Fessendonand Fessendon, (1982, Second Edition, Willard Grant Press, BostonMass.); Advanced Organic Chemistry by March (Third Edition, 1985, Wileyand Sons, New York); and Advanced Organic Chemistry by Carey andSandberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).See, also, U.S. Pat. Nos. 7,045,337 and 7,083,970, which areincorporated by reference herein. In addition to unnatural amino acidsthat contain novel side chains, unnatural amino acids that may besuitable for use in the present invention also optionally comprisemodified backbone structures, including but not limited to, asillustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C6-C20 straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, a-hydroxy derivatives, g-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, a tri-O-acetyl-GlcNAcb-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the StaudingerPNAS 99:19-24, which is incorporated by reference herein, for additionalmethionine analogs. International Application No. PCT/US06/47822entitled “Compositions Containing, Methods Involving, and Uses ofNon-natural Amino Acids and Polypeptides,” which is incorporated byreference herein, describes reductive alkylation of an aromatic aminemoieties, including but not limited to, p-amino-phenylalanine andreductive amination.

In one embodiment, compositions of insulin polypeptide that include anunnatural amino acid (such as p-(propargyloxy)-phenyalanine) areprovided. Various compositions comprising p-(propargyloxy)-phenyalanineand, including but not limited to, proteins and/or cells, are alsoprovided. In one aspect, a composition that includes thep-(propargyloxy)-phenyalanine unnatural amino acid, further includes anorthogonal tRNA. The unnatural amino acid can be bonded (including butnot limited to, covalently) to the orthogonal tRNA, including but notlimited to, covalently bonded to the orthogonal tRNA though anamino-acyl bond, covalently bonded to a 3′OH or a 2′OH of a terminalribose sugar of the orthogonal tRNA, etc.

The chemical moieties via unnatural amino acids that can be incorporatedinto proteins offer a variety of advantages and manipulations of theprotein. For example, the unique reactivity of a keto functional groupallows selective modification of proteins with any of a number ofhydrazine- or hydroxylamine-containing reagents in vitro and in vivo. Aheavy atom unnatural amino acid, for example, can be useful for phasingX-ray structure data. The site-specific introduction of heavy atomsusing unnatural amino acids also provides selectivity and flexibility inchoosing positions for heavy atoms. Photoreactive unnatural amino acids(including but not limited to, amino acids with benzophenone andarylazides (including but not limited to, phenylazide) side chains), forexample, allow for efficient in vivo and in vitro photocrosslinking ofprotein. Examples of photoreactive unnatural amino acids include, butare not limited to, p-azido-phenylalanine and p-benzoyl-phenylalanine.The protein with the photoreactive unnatural amino acids can then becrosslinked at will by excitation of the photoreactive group-providingtemporal control. In one example, the methyl group of an unnatural aminocan be substituted with an isotopically labeled, including but notlimited to, methyl group, as a probe of local structure and dynamics,including but not limited to, with the use of nuclear magnetic resonanceand vibrational spectroscopy. Alkynyl or azido functional groups, forexample, allow the selective modification of proteins with moleculesthrough a [3+2] cycloaddition reaction.

A non-natural amino acid incorporated into a polypeptide at the aminoterminus can be composed of an R group that is any substituent otherthan one used in the twenty natural amino acids and a 2nd reactive groupdifferent from the NH2 group normally present in α-amino acids (seeFormula I). A similar non-natural amino acid can be incorporated at thecarboxyl terminus with a 2nd reactive group different from the COOHgroup normally present in α-amino acids (see Formula I).

The unnatural amino acids of the invention may be selected or designedto provide additional characteristics unavailable in the twenty naturalamino acids. For example, unnatural amino acid may be optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties may be optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

Structure and Synthesis of Non-Natural Amino Acids: Carbonyl,Carbonyl-Like, Masked Carbonyl, Protected Carbonyl Groups, andHydroxylamine Groups

In some embodiments the present invention provides insulin linked to awater soluble polymer, e.g., a PEG, by an oxime bond.

Many types of non-naturally encoded amino acids are suitable forformation of oxime bonds. These include, but are not limited to,non-naturally encoded amino acids containing a carbonyl, dicarbonyl, orhydroxylamine group. Such amino acids are described in U.S. PatentPublication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 and WO2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” which areincorporated herein by reference in their entirety. Non-naturallyencoded amino acids are also described in U.S. Pat. No. 7,083,970 andU.S. Pat. No. 7,045,337, which are incorporated by reference herein intheir entirety.

Some embodiments of the invention utilize insulin polypeptides that aresubstituted at one or more positions with a para-acetylphenylalanineamino acid. The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine are described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), incorporated by reference. Othercarbonyl- or dicarbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art. Further, non-limiting examplarysyntheses of non-natural amino acid that are included herein arepresented in FIGS. 4, 24-34 and 36-39 of U.S. Pat. No. 7,083,970, whichis incorporated by reference herein in its entirety.

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via nucleophilic addition reactions amongothers. Such electrophilic reactive groups include a carbonyl group(including a keto group and a dicarbonyl group), a carbonyl-like group(which has reactivity similar to a carbonyl group (including a ketogroup and a dicarbonyl group) and is structurally similar to a carbonylgroup), a masked carbonyl group (which can be readily converted into acarbonyl group (including a keto group and a dicarbonyl group)), or aprotected carbonyl group (which has reactivity similar to a carbonylgroup (including a keto group and a dicarbonyl group) upondeprotection). Such amino acids include amino acids having the structureof Formula (IV):

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)-, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)-, —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-,—N(R′)S(O)_(k)N(R′)-, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;

J is

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;each R″ is independently H, alkyl, substituted alkyl, or a protectinggroup, or when more thanone R″ group is present, two R″ optionally form a heterocycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each of R₃ and R₄ is independently H, halogen, lower alkyl, orsubstituted lower alkyl, or R₃ and R₄ or two R₃ groups optionally form acycloalkyl or a heterocycloalkyl;or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkylor heterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group;or the -J-R group together forms a monocyclic or bicyclic cycloalkyl orheterocycloalkyl comprising at least one carbonyl group, including adicarbonyl group, protected carbonyl group, including a protecteddicarbonyl group, or masked carbonyl group, including a maskeddicarbonyl group;with a proviso that when A is phenylene and each. R₃ is H, B is present;and that when A is —(CH₂)₄— and each R₃ is B is not —NHC(O)(CH₂CH₂)—;and that when A and B are absent and each R₃ is H, R is not methyl.

In addition, having the structure of Formula (V) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)-, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)-, —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-,—N(R′)S(O)_(k)N(R′)-, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;with a proviso that when A is phenylene, B is present; and that when Ais —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; and that when A and B areabsent, R is not methyl.

In addition, amino acids having the structure of Formula (VI) areincluded:

wherein:B is a linker selected from the group consisting of lower alkylene,substituted lower alkylene, lower alkenylene, substituted loweralkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—,—O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substitutedalkylene)-, —S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene orsubstituted alkylene)-, —C(O)—, —C(O)-(alkylene or substitutedalkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)-,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)-,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO—(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected group, carboxylprotected or a salt thereof. In addition, any of the followingnon-natural amino acids may be incorporated into a non-natural aminoacid polypeptide.

In addition, the following amino acids having the structure of Formula(VII) are included:

whereinB is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8;with a proviso that when A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(VIII) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkenylene, substitutedalkenylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)-, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)-, —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-,—N(R′)S(O)_(k)N(R′)-, —N(R′)-13 N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)-, where each R′ isindependently H, alkyl, or substituted alkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(IX) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)-, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)-, —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-,—N(R′)S(O)_(k)N(R′)-, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;wherein each R_(a) is independently selected from the group consistingof H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ isindependently H, alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(X) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)-, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)-, —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-,—N(R′)S(O)_(k)N(R′)-, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)-,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)-, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each Ra is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)2, —C(O)kR′ where k is 1, 2, or3, —C(O)N(R′)2, —OR′, and —S(O)kR′, where each R′ is independently H,alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids having the structure of Formula(XI) are included:

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, O-(alkylene or substitutedalkylene)-, —S—, S-(alkylene or substituted alkylene)-, —S(O)k—where kis 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-, C(O)—,C(O)-(alkylene or substituted alkylene)-, —C(S)—, C(S)-(alkylene orsubstituted alkylene)-, —N(R′)-, NR′-(alkylene or substitutedalkylene)-, C(O)N(R′)-, CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)-, CSN(R′)-(alkylene or substituted alkylene)-,N(R′)CO-(alkylene or substituted alkylene)-, N(R′)C(O)O—, S(O)kN(R′)-,N(R′)C(O)N(R′)-, N(R′)C(S)N(R′)-, N(R′)S(O)kN(R′)-, N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)-, —C(R′)═N—N═, C(R′)2-N═N—, and C(R′)2N(R′)N(R′)-, whereeach R′ is independently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, the following amino acids having the structure of Formula(XII) are included:

B is optional, and when present is a linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)-, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)-, —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-,—N(R′)S(O)_(k)N(R′)-, —N(R′)—N—, —C(R′)═N—, —C(R′)═N—N(R′)-,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)-, where each R′ isindependently H, alkyl, or substituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;wherein each Ra is independently selected from the group consisting ofH, halogen, alkyl, substituted alkyl, —N(R′)2, —C(O)kR′ where k is 1, 2,or 3, —C(O)N(R′)2, —OR′, and —S(O)kR′, where each R′ is independently H,alkyl, or substituted alkyl.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIII) are included:

wherein B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)-, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)-, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)-, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)-, —N(R′)C(O)N(R′)-, —N(R′)C(S)N(R′)-,—N(R′)S(O)_(k)N(R′)-, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N═, —C(R′)₂—N═N—,and —C(R′)₂—N(R′)—N(R′)-, where each R′ is independently H, alkyl, orsubstituted alkyl;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;

each R_(a) is independently selected from the group consisting H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl; and n is 0 to 8.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof. In addition, these non-natural amino acids and any ofthe following non-natural amino acids may be incorporated into anon-natural amino acid polypeptide.

In addition, the following amino acids having the structure of Formula(XIV) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X1 is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XIV-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R1 is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR2 is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ oneach CR⁸R⁹ group is independently selected from the group consisting ofH, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ cantogether form ═O or a cycloalkyl, or any to adjacent R⁸ groups cantogether form a cycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R⁸ groups can together form acycloalkyl.

In addition, the following amino acids having the structure of Formula(XV-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;n is 0, 1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of alkoxy, alkylamine,halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═O or acycloalkyl, or any to adjacent R⁸ groups can together form a cycloalkyl,

In addition, the following amino acids having the structure of Formula(XVI) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;X₁ is C, S, or S(O); and L is alkylene, substituted alkylene,N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-A) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having the structure of Formula(XVI-B) are included:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;L is alkylene, substituted alkylene, N(R′)(alkylene) orN(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, amino acids having the structure of Formula (XVII) areincluded:

wherein:A is optional, and when present is lower alkylene, substituted loweralkylene, lower cycloalkylene, substituted lower cycloalkylene, loweralkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide.

In addition, amino acids having the structure of Formula (XVIII) areincluded:

wherein:

M is —C(R₃)—,

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl;R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl;T₃ is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl;R₁ is optional, and when present, is H, an amino protecting group,resin, amino acid, polypeptide, or polynucleotide; andR₂ is optional, and when present, is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide;each R_(a) is independently selected from the group consisting of H,halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2,or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independentlyH, alkyl, or substituted alkyl.

In addition, amino acids having the structure of Formula (XIX) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; and

T₃ is O, or S.

In addition, amino acids having the structure of Formula (XX) areincluded:

wherein:R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl.

In addition, the following amino acids having structures of Formula(XXI) are included:

In some embodiments, a polypeptide comprising a non-natural amino acidis chemically modified to generate a reactive carbonyl or dicarbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et. al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-natural amino acid bearing adjacenthydroxyl and amino groups can be incorporated into the polypeptide as a“masked” aldehyde functionality. For example, 5-hydroxylysine bears ahydroxyl group adjacent to the epsilon amine. Reaction conditions forgenerating the aldehyde typically involve addition of molar excess ofsodium metaperiodate under mild conditions to avoid oxidation at othersites within the polypeptide. The pH of the oxidation reaction istypically about 7.0. A typical reaction involves the addition of about1.5 molar excess of sodium meta periodate to a buffered solution of thepolypeptide, followed by incubation for about 10 minutes in the dark.See, e.g. U.S. Pat. No. 6,423,685.

The carbonyl or dicarbonyl functionality can be reacted selectively witha hydroxylamine-containing reagent under mild conditions in aqueoussolution to form the corresponding oxime linkage that is stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonyl ordicarbonyl group allows for selective modification in the presence ofthe other amino acid side chains. See, e.g., Cornish, V. W., et al., J.Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G.,Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., Science276:1125-1128 (1997).

Structure and Synthesis of Non-Natural Amino Acids:Hydroxylamine-Containing Amino Acids

U.S. patent application Ser. No. 11/316,534 (U.S. Publication No.20060189529) is incorporated by reference in its entirety. Thus, thedisclosures provided in Section V (entitled “Non-natural Amino Acids”),Part B (entitled “Structure and Synthesis of Non-Natural Amino AcidsHydroxylamine-Containing Amino Acids”), in U.S. patent application Ser.No. 11/316,534 (U.S. Publication No. 20060189529) apply fully to themethods, compositions (including Formulas I-XXXV), techniques andstrategies for making, purifying, characterizing, and using non-naturalamino acids, non-natural amino acid polypeptides and modifiednon-natural amino acid polypeptides described herein to the same extentas if such disclosures were fully presented herein. U.S. PatentPublication Nos. 2006/0194256, 2006/0217532, and 2006/0217289 and WO2006/069246 entitled “Compositions containing, methods involving, anduses of non-natural amino acids and polypeptides,” are also incorporatedherein by reference in their entirety.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of ordinary skillin the art. For organic synthesis techniques, see, e.g., OrganicChemistry by Fessendon and Fessendon, (1982, Second Edition, WillardGrant Press, Boston Mass.); Advanced Organic Chemistry by March (ThirdEdition, 1985, Wiley and Sons, New York); and Advanced Organic Chemistryby Carey and Sundberg (Third Edition, Parts A and B, 1990, Plenum Press,New York). Additional publications describing the synthesis of unnaturalamino acids include, e.g., WO 2002/085923 entitled “In vivoincorporation of Unnatural Amino Acids;” Matsoukas et al., (1995) J.Med. Chem., 38, 4660-4669; King, F. E. & Kidd, D. A. A. (1949) A NewSynthesis of Glutamine and of γ-Dipeptides of Glutamic Acid fromPhthylated Intermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. &Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as ModelSubstrates for Anti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752;Craig, J. C. et al. (1988) Absolute Configuration of the Enantiomers of7-Chloro-4 [[4-(diethylamino)-1-methylbutyl]amino]quinoline(Chloroquine). J. Org. Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. &Frappier, F. (1991) Glutamine analogues as Potential Antimalarials, Eur.J. Med. Chem. 26, 201-5; Koskinen, A. M. P. & Rapoport, H. (1989)Synthesis of 4-Substituted Prolines as Conformationally ConstrainedAmino Acid Analogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. &Rapoport, H. (1985) Synthesis of Optically Pure Pipecolates fromL-Asparagine. Application to the Total Synthesis of (+)-Apovincaminethrough Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem. 50:1239-1246; Barton et al., (1987) Synthesis of Novelalpha-Amino-Acids and Derivatives Using Radical Chemistry: Synthesis ofL- and D-alpha-Amino-Adipic Acids, L-alpha-aminopimelic Acid andAppropriate Unsaturated Derivatives. Tetrahedron 43:4297-4308; and,Subasinghe et al., (1992) Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35:4602-7. Seealso, U.S. Patent Publication No. US 2004/0198637 entitled “ProteinArrays,” which is incorporated by reference herein.

A. Carbonyl Reactive Groups

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

The synthesis of p-acetyl-(+/−)-phenylalanine andm-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al.,Biochemistry 42: 6735-6746 (2003), which is incorporated by referenceherein. Other carbonyl-containing amino acids can be similarly preparedby one of ordinary skill in the art.

In some embodiments, a polypeptide comprising a non-naturally encodedamino acid is chemically modified to generate a reactive carbonylfunctional group. For instance, an aldehyde functionality useful forconjugation reactions can be generated from a functionality havingadjacent amino and hydroxyl groups. Where the biologically activemolecule is a polypeptide, for example, an N-terminal serine orthreonine (which may be normally present or may be exposed via chemicalor enzymatic digestion) can be used to generate an aldehydefunctionality under mild oxidative cleavage conditions using periodate.See, e.g., Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992);Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertneret al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known inthe art are restricted to the amino acid at the N-terminus of thepeptide or protein.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

B. Hydrazine, Hydrazide or Semicarbazide Reactive Groups

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-□-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one of ordinary skill in theart. See, e.g., U.S. Pat. No. 6,281,211, which is incorporated byreference herein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

C. Aminooxy-Containing Amino Acids

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G., Life Sci.60: 1635-1641 (1997). Other aminooxy-containing amino acids can beprepared by one of ordinary skill in the art.

D. Azide and Alkyne Reactive Groups

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occuring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing insulinpolypeptide can be carried out at room temperature under aqueousconditions by the addition of Cu(II) (including but not limited to, inthe form of a catalytic amount of CuSO₄) in the presence of a reducingagent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See,e.g., Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe,C. W., et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al.,Angew, Chem. Int. Ed. 41:2596-2599 (2002). Exemplary reducing agentsinclude, including but not limited to, ascorbate, metallic copper,quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, andan applied electric potential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between anazide and an alkyne is desired, the insulin polypeptide comprises anon-naturally encoded amino acid comprising an alkyne moiety and thewater soluble polymer to be attached to the amino acid comprises anazide moiety. Alternatively, the converse reaction (i.e., with the azidemoiety on the amino acid and the alkyne moiety present on the watersoluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine).

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one of ordinary skill in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; in is 0-10; R₂ is H,an amino acid, a polypeptide, or an amino terminus modification group,and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe β-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofordinary skill in the art, including but not limited to, viadisplacement of a suitable leaving group (including but not limited to,halide, mesylate, tosylate) or via opening of a suitably protectedlactone. See, e.g., Advanced Organic Chemistry by March (Third Edition,1985, Wiley and Sons, New York).

E. Aminothiol Reactive Groups

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated into insulinpolypeptides and then reacted with water soluble polymers comprising analdehyde functionality. In some embodiments, a water soluble polymer,drug conjugate or other payload can be coupled to an insulin polypeptidecomprising a beta-substituted aminothiol amino acid via formation of thethiazolidine.

F. Additional Reactive Groups

Additional reactive groups and non-naturally encoded amino acids,including but not limited to para-amino-phenylalanine, that can beincorporated into insulin polypeptides of the invention are described inthe following patent applications which are all incorporated byreference in their entirety herein: U.S. Patent Publication No.2006/0194256, U.S. Patent Publication No. 2006/0217532, U.S. PatentPublication No. 2006/0217289, U.S. Provisional Patent No. 60/755,338;U.S. Provisional Patent No. 60/755,711; U.S. Provisional Patent No.60/755,018; International Patent Application No. PCT/US06/49397; WO2006/069246; U.S. Provisional Patent No. 60/743,041; U.S. ProvisionalPatent No. 60/743,040; International Patent Application No,PCT/US06147822; U.S. Provisional Patent No. 60/882,819; U.S. ProvisionalPatent No. 60/882,500; and U.S. Provisional Patent No. 60/870,594. Theseapplications also discuss reactive groups that may be present on PEG orother polymers, including but not limited to, hydroxylamine (aminooxy)groups for conjugation.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake by a cell is one issue that is typicallyconsidered when designing and selecting unnatural amino acids, includingbut not limited to, for incorporation into a protein. For example, thehigh charge density of α-amino acids suggests that these compounds areunlikely to be cell permeable. Natural amino acids are taken up into theeukaryotic cell via a collection of protein-based transport systems. Arapid screen can be done which assesses which unnatural amino acids, ifany, are taken up by cells. See, e.g., the toxicity assays in, e.g.,U.S. Patent Publication No. US 2004/0198637 entitled “Protein Arrays”which is incorporated by reference herein; and Liu, D. R. & Schultz, P.O. (1999) Progress toward the evolution of an organism with an expandedgenetic code. PNAS United States 96:4780-4785. Although uptake is easilyanalyzed with various assays, an alternative to designing unnaturalamino acids that are amenable to cellular uptake pathways is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, including butnot limited to, in a cell, the invention provides such methods. Forexample, biosynthetic pathways for unnatural amino acids are optionallygenerated in host cell by adding new enzymes or modifying existing hostcell pathways. Additional new enzymes are optionally naturally occurringenzymes or artificially evolved enzymes. For example, the biosynthesisof p-aminophenylalanine (as presented in an example in WO 2002/085923entitled “In vivo incorporation of unnatural amino acids”) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a eukaryotic cell bytransforming the cell with a plasmid comprising the genes. The genes,when expressed in the cell, provide an enzymatic pathway to synthesizethe desired compound. Examples of the types of enzymes that areoptionally added are provided in the examples below. Additional enzymessequences are found, for example, in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, including but not limited to, asdeveloped by Maxygen, Inc. (available on the World Wide Web atmaxygen.com), is optionally used to develop novel enzymes and pathways.See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNAshuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751.Similarly DesignPath™, developed by Genencor (available on the WorldWide Web at genencor.com) is optionally used for metabolic pathwayengineering, including but not limited to, to engineer a pathway tocreate O-methyl-L-tyrosine in a cell. This technology reconstructsexisting pathways in host organisms using a combination of new genes,including but not limited to, those identified through functionalgenomics, and molecular evolution and design. Diversa Corporation(available on the World Wide Web at diversa.com) also providestechnology for rapidly screening libraries of genes and gene pathways,including but not limited to, to create new pathways.

Typically, the unnatural amino acid produced with an engineeredbiosynthetic pathway of the invention is produced in a concentrationsufficient for efficient protein biosynthesis, including but not limitedto, a natural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a cell is transformed with a plasmid comprisingthe genes used to produce enzymes desired for a specific pathway and anunnatural amino acid is generated, in vivo selections are optionallyused to further optimize the production of the unnatural amino acid forboth ribosomal protein synthesis and cell growth.

Polypeptides with Unnatural Amino Acids

The incorporation of an unnatural amino acid can be done for a varietyof purposes, including but not limited to, tailoring changes in proteinstructure and/or function, changing size, acidity, nucleophilicity,hydrogen bonding, hydrophobicity, accessibility of protease targetsites, targeting to a moiety (including but not limited to, for aprotein array), adding a biologically active molecule, attaching apolymer, attaching a radionuclide, modulating serum half-life,modulating tissue penetration (e.g. tumors), modulating activetransport, modulating tissue, cell or organ specificity or distribution,modulating immunogenicity, modulating protease resistance, etc. Proteinsthat include an unnatural amino acid can have enhanced or even entirelynew catalytic or biophysical properties. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, structural properties,spectroscopic properties, chemical and/or photochemical properties,catalytic ability, half-life (including but not limited to, serumhalf-life), ability to react with other molecules, including but notlimited to, covalently or noncovalently, and the like. The compositionsincluding proteins that include at least one unnatural amino acid areuseful for, including but not limited to, novel therapeutics,diagnostics, catalytic enzymes, industrial enzymes, binding proteins(including but not limited to, antibodies), and including but notlimited to, the study of protein structure and function. See, e.g.,Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structureand Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the invention, a composition includes at least oneprotein with at least one, including but not limited to, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, or at least ten or more unnaturalamino acids. The unnatural amino acids can be the same or different,including but not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more different sites in the protein that comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 or more different unnatural amino acids. In anotheraspect, a composition includes a protein with at least one, but fewerthan all, of a particular amino acid present in the protein issubstituted with the unnatural amino acid. For a given protein with morethan one unnatural amino acids, the unnatural amino acids can beidentical or different (including but not limited to, the protein caninclude two or more different types of unnatural amino acids, or caninclude two of the same unnatural amino acid). For a given protein withmore than two unnatural amino acids, the unnatural amino acids can bethe same, different or a combination of a multiple unnatural amino acidof the same kind with at least one different unnatural amino acid.

Proteins or polypeptides of interest with at least one unnatural aminoacid are a feature of the invention. The invention also includespolypeptides or proteins with at least one unnatural amino acid producedusing the compositions and methods of the invention. An excipient(including but not limited to, a pharmaceutically acceptable excipient)can also be present with the protein.

By producing proteins or polypeptides of interest with at least oneunnatural amino acid in eukaryotic cells, proteins or polypeptides willtypically include eukaryotic post-translational modifications. Incertain embodiments, a protein includes at least one unnatural aminoacid and at least one post-translational modification that is made invivo by a eukaryotic cell, where the post-translational modification isnot made by a prokaryotic cell. For example, the post-translationmodification includes, including but not limited to, acetylation,acylation, lipid-modification, palmitoylation, palmitate addition,phosphorylation, glycolipid-linkage modification, glycosylation, and thelike. In one aspect, the post-translational modification includesattachment of an oligosaccharide (including but not limited to,(GlcNAc-Man)2-Man-GlcNAc-GlcNAc)) to an asparagine by aGlcNAc-asparagine linkage. Sec Table 1 which lists some examples ofN-linked oligosaccharides of eukaryotic proteins (additional residuescan also be present, which are not shown). In another aspect, thepost-translational modification includes attachment of anoligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc,etc.) to a serine or threonine by a GalNAc-serine or GalNAc-threoninelinkage, or a GlcNAc-serine or a GlcNAc-threonine linkage.

TABLE 1 Examples of oligosaccharides through GLCNAC-linkage Type BaseStructure HIGH-MANNOSE

HYBRID

COMPLEX

XYLOSE

In yet another aspect, the post-translation modification includesproteolytic processing of precursors (including but not limited to,calcitonin precursor, calcitonin gene-related peptide precursor,preproparathyroid hormone, preproinsulin, proinsulin,prepro-opiomelanocortin, pro-opiomelanocortin and the like), assemblyinto a multisubunit protein or macromolecular assembly, translation toanother site in the cell (including but not limited to, to organelles,such as the endoplasmic reticulum, the Golgi apparatus, the nucleus,lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., orthrough the secretory pathway). In certain embodiments, the proteincomprises a secretion or localization sequence, an epitope tag, a FLAGtag, a polyhistidine tag, a GST fusion, or the like.

One advantage of an unnatural amino acid is that it presents additionalchemical moieties that can be used to add additional molecules. Thesemodifications can be made in vivo in a eukaryotic or non-eukaryoticcell, or in vitro. Thus, in certain embodiments, the post-translationalmodification is through the unnatural amino acid. For example, thepost-translational modification can be through anucleophilic-electrophilic reaction. Most reactions currently used forthe selective modification of proteins involve covalent bond formationbetween nucleophilic and electrophilic reaction partners, including butnot limited to the reaction of α-haloketones with histidine or cysteineside chains. Selectivity in these cases is determined by the number andaccessibility of the nucleophilic residues in the protein. In proteinsof the invention, other more selective reactions can be used such as thereaction of an unnatural keto-amino acid with hydrazides or aminooxycompounds, in vitro and in vivo. See, e.g., Cornish, et al., (1996) J.Am. Chem. Soc., 118:8150-8151; Mahal, et al., (1997) Science,276:1125-1128; Wang, et al., (2001) Science 292:498-500; Chin, et al.,(2002) J. Am. Chem. Soc. 124:9026-9027; Chin, et al., (2002) Proc. Natl.Acad. Sci., 99:11020-11024; Wang, et al., (2003) Proc. Natl. Acad. Sci.,100:56-61; Zhang, et al., (2003) Biochemistry, 42:6735-6746; and, Chin,et al., (2003) Science, 301:964-7, all of which are incorporated byreference herein. This allows the selective labeling of virtually anyprotein with a host of reagents including fluorophores, crosslinkingagents, saccharide derivatives and cytotoxic molecules. See also, U.S.Pat. No. 6,927,042 entitled “Glycoprotein synthesis,” which isincorporated by reference herein. Post-translational modifications,including but not limited to, through an azido amino acid, can also madethrough the Staudinger ligation (including but not limited to, withtriarylphosphine reagents). See, e.g., Kiick et al., (2002)Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligation, PNAS 99:19-24.

This invention provides another highly efficient method for theselective modification of proteins, which involves the geneticincorporation of unnatural amino acids, including but not limited to,containing an azide or alkynyl moiety into proteins in response to aselector codon. These amino acid side chains can then be modified by,including but not limited to, a Huisgen [3+2] cycloaddition reaction(see, e.g., Padwa, A. in Comprehensive Organic Synthesis, Vol. 4, (1991)Ed. Trost, B. M., Pergamon, Oxford, p. 1069-1109; and, Huisgen, R. in1,3-Dipolar Cycloaddition Chemistry, (1984) Ed. Padwa, A., Wiley, NewYork, p. 1-176) with, including but not limited to, alkynyl or azidederivatives, respectively. Because this method involves a cycloadditionrather than a nucleophilic substitution, proteins can be modified withextremely high selectivity. This reaction can be carried out at roomtemperature in aqueous conditions with excellent regioselectivity(1,4>1,5) by the addition of catalytic amounts of Cu(I) salts to thereaction mixture. See, e.g., Tornoe, et al., (2002) J. Org. Chem.67:3057-3064; and, Rostovtsev, et al., (2002) Angew. Chem. Int. Ed.41:2596-2599. Another method that can be used is the ligand exchange ona bisarsenic compound with a tetracysteine motif, see, e.g., Griffin, etal., (1998) Science 281:269-272.

A molecule that can be added to a protein of the invention through a[3+2] cycloaddition includes virtually any molecule with an azide oralkynyl derivative. Molecules include, but are not limited to, dyes,fluorophores, crosslinking agents, saccharide derivatives, polymers(including but not limited to, derivatives of polyethylene glycol),photocrosslinkers, cytotoxic compounds, affinity labels, derivatives ofbiotin, resins, beads, a second protein or polypeptide (or more),polynucleotide(s) (including but not limited to, DNA, RNA, etc.), metalchelators, cofactors, fatty acids, carbohydrates, and the like. Thesemolecules can be added to an unnatural amino acid with an alkynyl group,including but not limited to, p-propargyloxyphenylalanine, or azidogroup, including but not limited to, p-azido-phenyl alanine,respectively.

In Vivo Generation of Insulin Polypeptides ComprisingNon-Naturally-Encoded Amino Acids

The insulin polypeptides of the invention can be generated in vivo usingmodified tRNA and tRNA synthetases to add to or substitute amino acidsthat are not encoded in naturally-occurring systems.

Methods for generating tRNAs and tRNA synthetases which use amino acidsthat are not encoded in naturally-occurring systems are described in,e.g., U.S. Pat. Nos. 7,045,337 and 7,083,970 which are incorporated byreference herein. These methods involve generating a translationalmachinery that functions independently of the synthetases and tRNAsendogenous to the translation system (and are therefore sometimesreferred to as “orthogonal”). Typically, the translation systemcomprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNAsynthetase (O-RS). Typically, the O-RS preferentially aminoacylates theO-tRNA with at least one non-naturally occurring amino acid in thetranslation system and the O-tRNA recognizes at least one selector codonthat is not recognized by other tRNAs in the system. The translationsystem thus inserts the non-naturally-encoded amino acid into a proteinproduced in the system, in response to an encoded selector codon,thereby “substituting” an amino acid into a position in the encodedpolypeptide.

A wide variety of orthogonal tRNAs and aminoacyl tRNA synthetases havebeen described in the art for inserting particular synthetic amino acidsinto polypeptides, and are generally suitable for use in the presentinvention. For example, keto-specific O-tRNA/aminoacyl-tRNA synthetasesare described in Wang, L., et al., Proc. Natl. Acad. Sci. USA 100:56-61(2003) and Zhang, Z. et al., Biochem. 42(22):6735-6746 (2003). ExemplaryO-RS, or portions thereof, are encoded by polynucleotide sequences andinclude amino acid sequences disclosed in U.S. Pat. Nos. 7,045,337 and7,083,970, each incorporated herein by reference. Corresponding O-tRNAmolecules for use with the O-RSs are also described in U.S. Pat. Nos.7,045,337 and 7,083,970 which are incorporated by reference herein.Additional examples of O-tRNA/aminoacyl-tRNA synthetase pairs aredescribed in WO 2005/007870, WO 2005/007624; and WO 2005/019415.

An example of an azide-specific O-tRNA/aminoacyl-tRNA synthetase systemis described in Chin, J. W., et al., J. Am. Chem. Soc. 124:9026-9027(2002). Exemplary O—RS sequences for p-azido-L-Phe include, but are notlimited to, nucleotide sequences SEQ ID NOs: 14-16 and 29-32 and aminoacid sequences SEQ ID NOs: 46-48 and 61-64 as disclosed in U.S. Pat. No.7,083,970 which is incorporated by reference herein. Exemplary O-tRNAsequences suitable for use in the present invention include, but are notlimited to, nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S.Pat. No. 7,083,970, which is incorporated by reference herein. Otherexamples of O-tRNA/aminoacyl-tRNA synthetase pairs specific toparticular non-naturally encoded amino acids are described in U.S. Pat.No. 7,045,337 which is incorporated by reference herein. O-RS and O-tRNAthat incorporate both keto- and azide-containing amino acids in S.cerevisiae are described in Chin, J. W., et al., Science 301:964-967(2003).

Several other orthogonal pairs have been reported. Glutaminyl (see,e.g., Liu, D. R., and Schultz, P. G. (1999) Proc. Natl. Acad. Sci.U.S.A. 96:4780-4785), aspartyl (see, e.g., Pastrnak, M., et al., (2000)Helv. Chim. Acta 83:2277-2286), and tyrosyl (see, e.g., Ohno, S., etal., (1998) J. Biochem. (Tokyo, Jpn.) 124:1065-1068; and, Kowal, A. K.,et al., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) systemsderived from S. cerevisiae tRNA's and synthetases have been describedfor the potential incorporation of unnatural amino acids in E. coli.Systems derived from the E. coli glutaminyl (see, e.g., Kowal, A. K., etal., (2001) Proc. Natl. Acad. Sci. U.S.A. 98:2268-2273) and tyrosyl(see, e.g., Edwards, H., and Schimmel, P. (1990) Mol. Cell. Biol.10:1633-1641) synthetases have been described for use in S. cerevisiae.The E. coli tyrosyl system has been used for the incorporation of3-iodo-L-tyrosine in vivo, in mammalian cells. See, Sakamoto, K., etal., (2002) Nucleic Acids Res. 30:4692-4699.

Use of O-tRNA/aminoacyl-tRNA synthetases involves selection of aspecific codon which encodes the non-naturally encoded amino acid. Whileany codon can be used, it is generally desirable to select a codon thatis rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNAsynthetase is expressed. For example, exemplary codons include nonsensecodon such as stop codons (amber, ochre, and opal), four or more basecodons and other natural three-base codons that are rarely or unused.

Specific selector codon(s) can be introduced into appropriate positionsin the insulin polynucleotide coding sequence using mutagenesis methodsknown in the art (including but not limited to, site-specificmutagenesis, cassette mutagenesis, restriction selection mutagenesis,etc.).

Methods for generating components of the protein biosynthetic machinery,such as O-RSs, O-tRNAs, and orthogonal O-tRNA/O-RS pairs that can beused to incorporate a non-naturally encoded amino acid are described inWang, L., et al., Science 292: 498-500 (2001); Chin, J. W., et al., J.Am. Chem. Soc. 124:9026-9027 (2002); Zhang, Z. et al., Biochemistry 42:6735-6746 (2003). Methods and compositions for the in vivo incorporationof non-naturally encoded amino acids are described in U.S. Pat. No.7,045,337, which is incorporated by reference herein. Methods forselecting an orthogonal tRNA-tRNA synthetase pair for use in in vivotranslation system of an organism are also described in U.S. Pat. Nos.7,045,337 and 7,083,970 which are incorporated by reference herein. PCTPublication No. WO 04/035743 entitled “Site Specific Incorporation ofKeto Amino Acids into Proteins,” which is incorporated by referenceherein in its entirety, describes orthogonal RS and tRNA pairs for theincorporation of keto amino acids. PCT Publication No. WO 04/094593entitled “Expanding the Eukaryotic Genetic Code,” which is incorporatedby reference herein in its entirety, describes orthogonal RS and tRNApairs for the incorporation of non-naturally encoded amino acids ineukaryotic host cells.

Methods for producing at least one recombinant orthogonal aminoacyl-tRNAsynthetase (O-RS) comprise: (a) generating a library of (optionallymutant) RSs derived from at least one aminoacyl-tRNA synthetase (RS)from a first organism, including but not limited to, a prokaryoticorganism, such as Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, or the like, or aeukaryotic organism; (b) selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that aminoacylate an orthogonal tRNA(O-tRNA) in the presence of a non-naturally encoded amino acid and anatural amino acid, thereby providing a pool of active (optionallymutant) RSs; and/or, (c) selecting (optionally through negativeselection) the pool for active RSs (including but not limited to, mutantRSs) that preferentially aminoacylate the O-tRNA in the absence of thenon-naturally encoded amino acid, thereby providing the at least onerecombinant O-RS; wherein the at least one recombinant O-RSpreferentially aminoacylates the O-tRNA with the non-naturally encodedamino acid.

In one embodiment, the RS is an inactive RS. The inactive RS can begenerated by mutating an active RS. For example, the inactive RS can begenerated by mutating at least about 1, at least about 2, at least about3, at least about 4, at least about 5, at least about 6, or at leastabout 10 or more amino acids to different amino acids, including but notlimited to, alanine.

Libraries of mutant RSs can be generated using various techniques knownin the art, including but not limited to rational design based onprotein three dimensional RS structure, or mutagenesis of RS nucleotidesin a random or rational design technique. For example, the mutant RSscan be generated by site-specific mutations, random mutations, diversitygenerating recombination mutations, chimeric constructs, rational designand by other methods described herein or known in the art.

In one embodiment, selecting (and/or screening) the library of RSs(optionally mutant RSs) for members that are active, including but notlimited to, that aminoacylate an orthogonal tRNA (O-tRNA) in thepresence of a non-naturally encoded amino acid and a natural amino acid,includes: introducing a positive selection or screening marker,including but not limited to, an antibiotic resistance gene, or thelike, and the library of (optionally mutant) RSs into a plurality ofcells, wherein the positive selection and/or screening marker comprisesat least one selector codon, including but not limited to, an amber,ochre, or opal codon; growing the plurality of cells in the presence ofa selection agent; identifying cells that survive (or show a specificresponse) in the presence of the selection and/or screening agent bysuppressing the at least one selector codon in the positive selection orscreening marker, thereby providing a subset of positively selectedcells that contains the pool of active (optionally mutant) RSs.Optionally, the selection and/or screening agent concentration can bevaried.

In one aspect, the positive selection marker is a chloramphenicolacetyltransferase (CAT) gene and the selector codon is an amber stopcodon in the CAT gene. Optionally, the positive selection marker is aβ-lactamase gene and the selector codon is an amber stop codon in theβ-lactamase gene. In another aspect the positive screening markercomprises a fluorescent or luminescent screening marker or an affinitybased screening marker (including but not limited to, a cell surfacemarker).

In one embodiment, negatively selecting or screening the pool for activeRSs (optionally mutants) that preferentially aminoacylate the O-tRNA inthe absence of the non-naturally encoded amino acid includes:introducing a negative selection or screening marker with the pool ofactive (optionally mutant) RSs from the positive selection or screeninginto a plurality of cells of a second organism, wherein the negativeselection or screening marker comprises at least one selector codon(including but not limited to, an antibiotic resistance gene, includingbut not limited to, a chloramphenicol acetyltransferase (CAT) gene);and, identifying cells that survive or show a specific screeningresponse in a first medium supplemented with the non-naturally encodedamino acid and a screening or selection agent, but fail to survive or toshow the specific response in a second medium not supplemented with thenon-naturally encoded amino acid and the selection or screening agent,thereby providing surviving cells or screened cells with the at leastone recombinant O-RS. For example, a CAT identification protocoloptionally acts as a positive selection and/or a negative screening indetermination of appropriate O-RS recombinants. For instance, a pool ofclones is optionally replicated on growth plates containing CAT (whichcomprises at least one selector codon) either with or without one ormore non-naturally encoded amino acid. Colonies growing exclusively onthe plates containing non-naturally encoded amino acids are thusregarded as containing recombinant O-RS. In one aspect, theconcentration of the selection (and/or screening) agent is varied. Insome aspects the first and second organisms are different. Thus, thefirst and/or second organism optionally comprises: a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacterium, a eubacterium, a plant, an insect, a protist, etc. Inother embodiments, the screening marker comprises a fluorescent orluminescent screening marker or an affinity based screening marker.

In another embodiment, screening or selecting (including but not limitedto, negatively selecting) the pool for active (optionally mutant) RSsincludes: isolating the pool of active mutant RSs from the positiveselection step (b); introducing a negative selection or screeningmarker, wherein the negative selection or screening marker comprises atleast one selector codon (including but not limited to, a toxic markergene, including but not limited to, a ribonuclease barnase gene,comprising at least one selector codon), and the pool of active(optionally mutant) RSs into a plurality of cells of a second organism;and identifying cells that survive or show a specific screening responsein a first medium not supplemented with the non-naturally encoded aminoacid, but fail to survive or show a specific screening response in asecond medium supplemented with the non-naturally encoded amino acid,thereby providing surviving or screened cells with the at least onerecombinant O-RS, wherein the at least one recombinant O-RS is specificfor the non-naturally encoded amino acid. In one aspect, the at leastone selector codon comprises about two or more selector codons. Suchembodiments optionally can include wherein the at least one selectorcodon comprises two or more selector codons, and wherein the first andsecond organism are different (including but not limited to, eachorganism is optionally, including but not limited to, a prokaryote, aeukaryote, a mammal, an Escherichia coli, a fungi, a yeast, anarchaebacteria, a eubacteria, a plant, an insect, a protist, etc.).Also, some aspects include wherein the negative selection markercomprises a ribonuclease barnase gene (which comprises at least oneselector codon). Other aspects include wherein the screening markeroptionally comprises a fluorescent or luminescent screening marker or anaffinity based screening marker. In the embodiments herein, thescreenings and/or selections optionally include variation of thescreening and/or selection stringency.

In one embodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof O-RS (optionally mutated) derived from the at least one recombinantO-RS; and, (f) repeating steps (b) and (c) until a mutated O-RS isobtained that comprises an ability to preferentially aminoacylate theO-tRNA. Optionally, steps (d)-(f) are repeated, including but notlimited to, at least about two times. In one aspect, the second set ofmutated O-RS derived from at least one recombinant O-RS can be generatedby mutagenesis, including but not limited to, random mutagenesis,site-specific mutagenesis, recombination or a combination thereof.

The stringency of the selection/screening steps, including but notlimited to, the positive selection/screening step (b), the negativeselection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c), in the above-described methods,optionally includes varying the selection/screening stringency. Inanother embodiment, the positive selection/screening step (b), thenegative selection/screening step (c) or both the positive and negativeselection/screening steps (b) and (c) comprise using a reporter, whereinthe reporter is detected by fluorescence-activated cell sorting (FACS)or wherein the reporter is detected by luminescence. Optionally, thereporter is displayed on a cell surface, on a phage display or the likeand selected based upon affinity or catalytic activity involving thenon-naturally encoded amino acid or an analogue. In one embodiment, themutated synthetase is displayed on a cell surface, on a phage display orthe like.

Methods for producing a recombinant orthogonal tRNA (O-tRNA) include:(a) generating a library of mutant tRNAs derived from at least one tRNA,including but not limited to, a suppressor tRNA, from a first organism;(b) selecting (including but not limited to, negatively selecting) orscreening the library for (optionally mutant) tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase (RS) from a secondorganism in the absence of a RS from the first organism, therebyproviding a pool of tRNAs (optionally mutant); and, (c) selecting orscreening the pool of tRNAs (optionally mutant) for members that areaminoacylated by an introduced orthogonal RS (O-RS), thereby providingat least one recombinant O-tRNA; wherein the at least one recombinantO-tRNA recognizes a selector codon and is not efficiency recognized bythe RS from the second organism and is preferentially aminoacylated bythe O-RS. In some embodiments the at least one tRNA is a suppressor tRNAand/or comprises a unique three base codon of natural and/or unnaturalbases, or is a nonsense codon, a rare codon, an unnatural codon, a codoncomprising at least 4 bases, an amber codon, an ochre codon, or an opalstop codon. In one embodiment, the recombinant O-tRNA possesses animprovement of orthogonality. It will be appreciated that in someembodiments, O-tRNA is optionally imported into a first organism from asecond organism without the need for modification. In variousembodiments, the first and second organisms are either the same ordifferent and are optionally chosen from, including but not limited to,prokaryotes (including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Escherichia coli, Halobacterium,etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria,plants, insects, protists, etc. Additionally, the recombinant tRNA isoptionally aminoacylated by a non-naturally encoded amino acid, whereinthe non-naturally encoded amino acid is biosynthesized in vivo eithernaturally or through genetic manipulation. The non-naturally encodedamino acid is optionally added to a growth medium for at least the firstor second organism.

In one aspect, selecting (including but not limited to, negativelyselecting) or screening the library for (optionally mutant) tRNAs thatare aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes:introducing a toxic marker gene, wherein the toxic marker gene comprisesat least one of the selector codons (or a gene that leads to theproduction of a toxic or static agent or a gene essential to theorganism wherein such marker gene comprises at least one selector codon)and the library of (optionally mutant) tRNAs into a plurality of cellsfrom the second organism; and, selecting surviving cells, wherein thesurviving cells contain the pool of (optionally mutant) tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example,surviving cells can be selected by using a comparison ratio cell densityassay.

In another aspect, the toxic marker gene can include two or moreselector codons. In another embodiment of the methods, the toxic markergene is a ribonuclease barnase gene, where the ribonuclease barnase genecomprises at least one amber codon. Optionally, the ribonuclease barnasegene can include two or more amber codons.

In one embodiment, selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS) can include: introducing a positive selection orscreening marker gene, wherein the positive marker gene comprises a drugresistance gene (including but not limited to, □-lactamase gene,comprising at least one of the selector codons, such as at least oneamber stop codon) or a gene essential to the organism, or a gene thatleads to detoxification of a toxic agent, along with the O-RS, and thepool of (optionally mutant) tRNAs into a plurality of cells from thesecond organism; and, identifying surviving or screened cells grown inthe presence of a selection or screening agent, including but notlimited to, an antibiotic, thereby providing a pool of cells possessingthe at least one recombinant tRNA, where the at least one recombinanttRNA is aminoacylated by the O-RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection and/or screening agent is varied.

Methods for generating specific O-tRNA/O-RS pairs are provided. Methodsinclude: (a) generating a library of mutant tRNAs derived from at leastone tRNA from a first organism; (b) negatively selecting or screeningthe library for (optionally mutant) tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of (optionallymutant) tRNAs; (e) selecting or screening the pool of (optionallymutant) tRNAs for members that are aminoacylated by an introducedorthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA.The at least one recombinant O-tRNA recognizes a selector codon and isnot efficiency recognized by the RS from the second organism and ispreferentially aminoacylated by the O-RS. The method also includes (d)generating a library of (optionally mutant) RSs derived from at leastone aminoacyl-tRNA synthetase (RS) from a third organism; (e) selectingor screening the library of mutant RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anon-naturally encoded amino acid and a natural amino acid, therebyproviding a pool of active (optionally mutant) RSs; and, (f) negativelyselecting or screening the pool for active (optionally mutant) RSs thatpreferentially aminoacylate the at least one recombinant O-tRNA in theabsence of the non-naturally encoded amino acid, thereby providing theat least one specific O-tRNA/O —RS pair, wherein the at least onespecific O-tRNA/O-RS pair comprises at least one recombinant O-RS thatis specific for the non-naturally encoded amino acid and the at leastone recombinant O-tRNA. Specific O-tRNA/O-RS pairs produced by themethods are included. For example, the specific O-tRNA/O-RS pair caninclude, including but not limited to, a mutRNATyr-mutTyrRS pair, suchas a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, amutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like.Additionally, such methods include wherein the first and third organismare the same (including but not limited to, Methanococcus jannaschii).

Methods for selecting an orthogonal tRNA-tRNA synthetase pair for use inan in vivo translation system of a second organism are also included inthe present invention. The methods include: introducing a marker gene, atRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from afirst organism into a first set of cells from the second organism;introducing the marker gene and the tRNA into a duplicate cell set froma second organism; and, selecting for surviving cells in the first setthat fail to survive in the duplicate cell set or screening for cellsshowing a specific screening response that fail to give such response inthe duplicate cell set, wherein the first set and the duplicate cell setare grown in the presence of a selection or screening agent, wherein thesurviving or screened cells comprise the orthogonal tRNA-tRNA synthetasepair for use in the in the in vivo translation system of the secondorganism. In one embodiment, comparing and selecting or screeningincludes an in vivo complementation assay. The concentration of theselection or screening agent can be varied.

The organisms of the present invention comprise a variety of organismand a variety of combinations. For example, the first and the secondorganisms of the methods of the present invention can be the same ordifferent. In one embodiment, the organisms are optionally a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, P. furiosus, P. horikoshii, A. pernix, T. thermophilus, orthe like. Alternatively, the organisms optionally comprise a eukaryoticorganism, including but not limited to, plants (including but notlimited to, complex plants such as monocots, or dicots), algae,protists, fungi (including but not limited to, yeast, etc), animals(including but not limited to, mammals, insects, arthropods, etc.), orthe like. In another embodiment, the second organism is a prokaryoticorganism, including but not limited to, Methanococcus jannaschii,Methanobacterium thermoautotrophicum, Halobacterium, Escherichia coli,A. fulgidus, Halobacterium, P. furiosus, P. horikoshii, A. pernix, T.thermophilus, or the like. Alternatively, the second organism can be aeukaryotic organism, including but not limited to, a yeast, a animalcell, a plant cell, a fungus, a mammalian cell, or the like. In variousembodiments the first and second organisms are different.

Location of Non-Naturally-Occurring Amino Acids in Insulin Polypeptides

The present invention contemplates incorporation of one or morenon-naturally-occurring amino acids into insulin polypeptides. One ormore non-naturally-occurring amino acids may be incorporated at aparticular position which does not disrupt activity of the polypeptide.This can be achieved by making “conservative” substitutions, includingbut not limited to, substituting hydrophobic amino acids withhydrophobic amino acids, bulky amino acids for bulky amino acids,hydrophilic amino acids for hydrophilic amino acids and/or inserting thenon-naturally-occurring amino acid in a location that is not requiredfor activity.

A variety of biochemical and structural approaches can be employed toselect the desired sites for substitution with a non-naturally encodedamino acid within the Insulinpolypeptide. It is readily apparent tothose of ordinary skill in the art that any position of the polypeptidechain is suitable for selection to incorporate a non-naturally encodedamino acid, and selection may be based on rational design or by randomselection for any or no particular desired purpose. Selection of desiredsites may be for producing an insulin molecule having any desiredproperty or activity, including but not limited to, agonists,super-agonists, inverse agonists, antagonists, receptor bindingmodulators, receptor activity modulators, dimer or multimer formation,no change to activity or property compared to the native molecule, ormanipulating any physical or chemical property of the polypeptide suchas solubility, aggregation, or stability. For example, locations in thepolypeptide required for biological activity of insulin polypeptides canbe identified using point mutation analysis, alanine scanning,saturation mutagenesis and screening for biological activity, or homologscanning methods known in the art. Other methods can be used to identifyresidues for modification of insulin polypeptides include, but are notlimited to, sequence profiling (Bowie and Eisenberg, Science 253(5016):164-70, (1991)), rotamer library selections (Dahiyat and Mayo, ProteinSci 5(5): 895-903 (1996); Dahiyat and Mayo, Science 278(5335): 82-7(1997); Desjarlais and Handel, Protein Science 4: 2006-2018 (1995);Harbury et al, PNAS USA 92(18): 8408-8412 (1995); Kono et al., Proteins:Structure, Function and Genetics 19: 244-255 (1994); Hellinga andRichards, PNAS USA 91: 5803-5807 (1994)); and residue pair potentials(Jones, Protein Science 3: 567-574, (1994)), and rational design usingProtein Design. Automation® technology. (See U.S. Pat. Nos. 6,188,965;6,269,312; 6,403,312; WO98/47089, which are incorporated by reference).Residues that are critical for insulin bioactivity, residues that areinvolved with pharmaceutical stability, antibody epitopes, or receptorbinding residues may be mutated. U.S. Pat. Nos. 5,580,723; 5,834,250;6,013,478; 6,428,954; and 6,451,561, which are incorporated by referenceherein, describe methods for the systematic analysis of the structureand function of polypeptides such as insulin by identifying activedomains which influence the activity of the polypeptide with a targetsubstance. Residues other than those identified as critical tobiological activity by alanine or homolog scanning mutagenesis may begood candidates for substitution with a non-naturally encoded amino aciddepending on the desired activity sought for the polypeptide.Alternatively, the sites identified as critical to biological activitymay also be good candidates for substitution with a non-naturallyencoded amino acid, again depending on the desired activity sought forthe polypeptide. Another alternative would be to simply make serialsubstitutions in each position on the polypeptide chain with anon-naturally encoded amino acid and observe the effect on theactivities of the polypeptide. It is readily apparent to those ofordinary skill in the art that any means, technique, or method forselecting a position for substitution with a non-natural amino acid intoany polypeptide is suitable for use in the present invention.

The structure and activity of mutants of insulin polypeptides thatcontain deletions can also be examined to determine regions of theprotein that are likely to be tolerant of substitution with anon-naturally encoded amino acid. In a similar manner, proteasedigestion and monoclonal antibodies can be used to identify regions ofinsulin that are responsible for binding the insulin receptor. Onceresidues that are likely to be intolerant to substitution withnon-naturally encoded amino acids have been eliminated, the impact ofproposed substitutions at each of the remaining positions can beexamined. Models may be generated from the three-dimensional crystalstructures of other insulin family members and insulin receptors.Protein Data Bank (PDB, available on the World Wide Web at rcsb.org) isa centralized database containing three-dimensional structural data oflarge molecules of proteins and nucleic acids. Models may be madeinvestigating the secondary and tertiary structure of polypeptides, ifthree-dimensional structural data is not available. Thus, those ofordinary skill in the art can readily identify amino acid positions thatcan be substituted with non-naturally encoded amino acids.

In some embodiments, the insulin polypeptides of the invention compriseone or more non-naturally encoded amino acids positioned in a region ofthe protein that does not disrupt the structure of the polypeptide.

Exemplary residues of incorporation of a non-naturally encoded aminoacid may be those that are excluded from potential receptor bindingregions, may be fully or partially solvent exposed, have minimal or nohydrogen-bonding interactions with nearby residues, may be minimallyexposed to nearby reactive residues, may be on one or more of theexposed faces, may be a site or sites that are juxtaposed to a secondinsulin, or other molecule or fragment thereof, may be in regions thatare highly flexible, or structurally rigid, as predicted by thethree-dimensional, secondary, tertiary, or quaternary structure ofinsulin, bound or unbound to its receptor, or coupled or not coupled toanother biologically active molecule, or may modulate the conformationof the insulin itself or a dimer or multimer comprising one or moreinsulin, by altering the flexibility or rigidity of the completestructure as desired.

One of ordinary skill in the art recognizes that such analysis ofinsulin enables the determination of which amino acid residues aresurface exposed compared to amino acid residues that are buried withinthe tertiary structure of the protein. Therefore, it is an embodiment ofthe present invention to substitute a non-naturally encoded amino acidfor an amino acid that is a surface exposed residue.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulin: inthe A chain before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., atthe carboxyl terminus of the protein), and any combination thereof (SEQID NO: 1) or in the B chain before position 1 (i.e. at the N-terminus),1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 (SEQ ID NO: 2).

An examination of the crystal structure of insulin and its interactionwith the insulin receptor can indicate which certain amino acid residueshave side chains that are fully or partially accessible to solvent. Theside chain of a non-naturally encoded amino acid at these positions maypoint away from the protein surface and out into the solvent.

In some embodiments, the non-naturally encoded amino acid at one or moreof these positions is linked to a water soluble polymer, including butnot limited to, positions: in the A chain before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of the protein), andany combination thereof (SEQ ID NO: 1) or in the B chain before position1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31(SEQ ID NO: 2).

A wide variety of non-naturally encoded amino acids can be substitutedfor, or incorporated into, a given position in an insulin polypeptide.In general, a particular non-naturally encoded amino acid is selectedfor incorporation based on an examination of the three dimensionalcrystal structure of an insulin polypeptide or other insulin familymember or insulin analog with its receptor, a preference forconservative substitutions (i.e., aryl-based non-naturally encoded aminoacids, such as p-acetylphenylalanine or O-propargyltyrosine substitutingfor Phe, Tyr or Trp), and the specific conjugation chemistry that onedesires to introduce into the insulin polypeptide (e.g., theintroduction of 4-azidophenylalanine if one wants to effect a Huisgen[3+2] cycloaddition with a water soluble polymer bearing an alkynemoiety or a amide bond formation with a water soluble polymer that bearsan aryl ester that, in turn, incorporates a phosphine moiety)

In one embodiment, the method further includes incorporating into theprotein the unnatural amino acid, where the unnatural amino acidcomprises a first reactive group; and contacting the protein with amolecule (including but not limited to, a label, a dye, a polymer, awater-soluble polymer, a derivative of polyethylene glycol, aphotocrosslinker, a radionuclide, a cytotoxic compound, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a saccharide, a water-soluble dendrimer, cyclodextrin,an inhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, an actinicradiation excitable moiety, a photoisomerizable moiety, biotin, aderivative of biotin, a biotin analogue, a moiety incorporating a heavyatom, a chemically cleavable group, a photocleavable group, an elongatedside chain, a carbon-linked sugar, a redox-active agent, an aminothioacid, a toxic moiety, an isotopically labeled moiety, a biophysicalprobe, a phosphorescent group, a chemiluminescent group, an electrondense group, a magnetic group, an intercalating group, a chromophore, anenergy transfer agent, a biologically active agent, a detectable label,a small molecule, a quantum dot, a nanotransmitter, a radionucleotide,radiotransmitter, a neutron-capture agent, or any combination of theabove, or any other desirable compound or substance) that comprises asecond reactive group. The first reactive group reacts with the secondreactive group to attach the molecule to the unnatural amino acidthrough a [3+2] cycloaddition. In one embodiment, the first reactivegroup is an alkynyl or azido moiety and the second reactive group is anazido or alkynyl moiety. For example, the first reactive group is thealkynyl moiety (including but not limited to, in unnatural amino acidp-propargyloxyphenylalanine) and the second reactive group is the azidomoiety. In another example, the first reactive group is the azido moiety(including but not limited to, in the unnatural amino acidp-azido-L-phenylalanine) and the second reactive group is the alkynylmoiety.

In some cases, the non-naturally encoded amino acid substitution(s) willbe combined with other additions, substitutions or deletions within theinsulin polypeptide to affect other biological traits of the insulinpolypeptide. In some cases, the other additions, substitutions ordeletions may increase the stability (including but not limited to,resistance to proteolytic degradation) of the insulin polypeptide orincrease affinity of the insulin polypeptide for its receptor. In somecases, the other additions, substitutions or deletions may increase thepharmaceutical stability of the insulin polypeptide. In some cases, theother additions, substitutions or deletions may enhance the anti-viralactivity of the insulin polypeptide. In some cases, the other additions,substitutions or deletions may increase the solubility (including butnot limited to, when expressed in E. coli or other host cells) of theinsulin polypeptide. In some embodiments additions, substitutions ordeletions may increase the insulin polypeptide solubility followingexpression in E. coli or other recombinant host cells. In someembodiments sites are selected for substitution with a naturally encodedor non-natural amino acid in addition to another site for incorporationof a non-natural amino acid that results in increasing the polypeptidesolubility following expression in E. coli or other recombinant hostcells. In some embodiments, the insulin polypeptides comprise anotheraddition, substitution or deletion that modulates affinity for theinsulin polypeptide receptor, binding proteins, or associated ligand,modulates signal transduction after binding to the insulin receptor,modulates circulating half-life, modulates release or bio-availability,facilitates purification, or improves or alters a particular route ofadministration. In some embodiments, the insulin polypeptides comprisean addition, substitution or deletion that increases the affinity of theinsulin variant for its receptor. Similarly, insulin polypeptides cancomprise chemical or enzyme cleavage sequences, protease cleavagesequences, reactive groups, antibody-binding domains (including but notlimited to, FLAG or poly-His) or other affinity based sequences(including, but not limited to, FLAG, poly-His, GST, etc.) or linkedmolecules (including, but not limited to, biotin) that improve detection(including, but not limited to, GFP), purification, transport throughtissues or cell membranes, prodrug release or activation, insulin sizereduction, or other traits of the polypeptide.

In some embodiments, the substitution of a non-naturally encoded aminoacid generates an insulin antagonist. In some embodiments, anon-naturally encoded amino acid is substituted or added in a regioninvolved with receptor binding. In some embodiments, insulin antagonistscomprise at least one substitution that cause insulin to act as anantagonist. In some embodiments, the insulin antagonist comprises anon-naturally encoded amino acid linked to a water soluble polymer thatis present in a receptor binding region of the insulin molecule.

In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids aresubstituted with one or more non-naturally-encoded amino acids. In somecases, the insulin polypeptide further includes 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more substitutions of one or more non-naturally encoded aminoacids for naturally-occurring amino acids. For example, in someembodiments, one or more residues in insulin are substituted with one ormore non-naturally encoded amino acids. In some cases, the one or morenon-naturally encoded residues are linked to one or more lower molecularweight linear or branched PEGs, thereby enhancing binding affinity andcomparable serum half-life relative to the species attached to a single,higher molecular weight PEG.

In some embodiments, up to two of the following residues of insulin aresubstituted with one or more non-naturally-encoded amino acids.

Expression in Non-Eukaryotes and Eukaryotes

To obtain high level expression of a cloned insulin polynucleotide, onetypically subclones polynucleotides encoding an insulin polypeptide ofthe invention into an expression vector that contains a strong promoterto direct transcription, a transcription/translation terminator, and iffor a nucleic acid encoding a protein, a ribosome binding site fortranslational initiation. Suitable bacterial promoters are known tothose of ordinary skill in the art and described, e.g., in Sambrook etal. and Ausubel et al.

Bacterial expression systems for expressing insulin polypeptides of theinvention are available in, including but not limited to, E. coli,Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa,Pseudomonas putida, and Salmonella (Palva et al., Gene 22:229-235(1983); Mosbach et al., Nature 302:543-545 (1983)). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are known to thoseof ordinary skill in the art and are also commercially available. Incases where orthogonal tRNAs and aminoacyl tRNA synthetases (describedabove) are used to express the insulin polypeptides of the invention,host cells for expression are selected based on their ability to use theorthogonal components. Exemplary host cells include Gram-positivebacteria (including but not limited to B. brevis, B. subtilis, orStreptomyces) and Gram-negative bacteria (E. coli, Pseudomonasfluorescens, Pseudomonas aeruginosa, Pseudomonas putida), as well asyeast and other eukaryotic cells. Cells comprising O-tRNA/O-RS pairs canbe used as described herein.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to synthesize proteins that compriseunnatural amino acids in large useful quantities. In one aspect, thecomposition optionally includes, including but not limited to, at least10 micrograms, at least 50 micrograms, at least 75 micrograms, at least100 micrograms, at least 200 micrograms, at least 250 micrograms, atleast 500 micrograms, at least 1 milligram, at least 10 milligrams, atleast 100 milligrams, at least one gram, or more of the protein thatcomprises an unnatural amino acid, or an amount that can be achievedwith in vivo protein production methods (details on recombinant proteinproduction and purification are provided herein). In another aspect, theprotein is optionally present in the composition at a concentration of,including but not limited to, at least 10 micrograms of protein perliter, at least 50 micrograms of protein per liter, at least 75micrograms of protein per liter, at least 100 micrograms of protein perliter, at least 200 micrograms of protein per liter, at least 250micrograms of protein per liter, at least 500 micrograms of protein perliter, at least 1 milligram of protein per liter, or at least 10milligrams of protein per liter or more, in, including but not limitedto, a cell lysate, a buffer, a pharmaceutical buffer, or other liquidsuspension (including but not limited to, in a volume of, including butnot limited to, anywhere from about 1 nl to about 100 L or more). Theproduction of large quantities (including but not limited to, greaterthat that typically possible with other methods, including but notlimited to, in vitro translation) of a protein in a eukaryotic cellincluding at least one unnatural amino acid is a feature of theinvention.

A eukaryotic host cell or non-eukaryotic host cell of the presentinvention provides the ability to biosynthesize proteins that compriseunnatural amino acids in large useful quantities. For example, proteinscomprising an unnatural amino acid can be produced at a concentrationof, including but not limited to, at least 10 μg/liter, at least 50μg/liter, at least 75 μg/liter, at least 100 μg/liter, at least 200μg/liter, at least 250 μg/liter, or at least 500 μg/liter, at least 1mg/liter, at least 2 mg/liter, at least 3 mg/liter, at least 4 mg/liter,at least 5 mg/liter, at least 6 mg/liter, at least 7 mg/liter, at least8 mg/liter, at least 9 mg/liter, at least 10 mg/liter, at least 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900mg/liter, 1 g/liter, 5 g/liter, 10 g/liter or more of protein in a cellextract, cell lysate, culture medium, a buffer, and/or the like.

A number of vectors suitable for expression of insulin are commerciallyavailable. Useful expression vectors for eukaryotic hosts, include butare not limited to, vectors comprising expression control sequences fromSV40, bovine papilloma virus, adenovirus and cytomegalovirus. Suchvectors include pcDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) andpCI-neo (Stratagene, La Jolla, Calif., USA). Bacterial plasmids, such asplasmids from E. coli, including pBR322, pET3a and pET12a, wider hostrange plasmids, such as RP4, phage DNAs, e.g., the numerous derivativesof phage lambda, e.g., NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages may be used. The 2μ plasmid andderivatives thereof, the POT1 vector (U.S. Pat. No. 4,931,373 which isincorporated by reference), the pJSO37 vector described in (Okkels, Ann.New York Aced. Sci. 782, 202 207, 1996) and pPICZ A, B or C (Invitrogen)may be used with yeast host cells. For insect cells, the vectors includebut are not limited to, pVL941, pBG311 (Cate et al., “Isolation of theBovine and Human Genes for Mullerian Inhibiting Substance and Expressionof the Human Gene In Animal Cells”, Cell, 45, pp. 685 98 (1986),pBluebac 4.5 and pMelbac (Invitrogen, Carlsbad, Calif.).

The nucleotide sequence encoding an insulin polypeptide may or may notalso include sequence that encodes a signal peptide. The signal peptideis present when the polypeptide is to be secreted from the cells inwhich it is expressed. Such signal peptide may be any sequence. Thesignal peptide may be prokaryotic or eukaryotic. Coloma, M (1992) J.Imm. Methods 152:89 104) describe a signal peptide for use in mammaliancells (murine Ig kappa light chain signal peptide). Other signalpeptides include but are not limited to, the α-factor signal peptidefrom S. cerevisiae (U.S. Pat. No. 4,870,008 which is incorporated byreference herein), the signal peptide of mouse salivary amylase (O.Hagenbuchle et al., Nature 289, 1981, pp. 643-646), a modifiedcarboxypeptidase signal peptide (L. A. Valls et al., Cell 48, 1987, pp.887-897), the yeast BAR1 signal peptide (WO 87/02670, which isincorporated by reference herein), and the yeast aspartic protease 3(YAP3) signal peptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp.127-137).

Examples of suitable mammalian host cells are known to those of ordinaryskill in the art. Such host cells may be Chinese hamster ovary (CHO)cells, (e.g. CHO-K1; ATCC CCL-61), Green Monkey cells (COS) (e.g. COS 1(ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), BabyHamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), andhuman cells (e.g. HEK 293 (ATCC CRL-1573)), as well as plant cells intissue culture. These cell lines and others are available from publicdepositories such as the American Type Culture Collection, Rockville,Md. In order to provide improved glycosylation of the insulinpolypeptide, a mammalian host cell may be modified to expresssialyltransferase, e.g. 1,6-sialyltransferase, e.g. as described in U.S.Pat. No. 5,047,335, which is incorporated by reference herein.

Methods for the introduction of exogenous DNA into mammalian host cellsinclude but are not limited to, calcium phosphare-mediated transfection,electroporation, DEAE-dextran mediated transfection, liposome-mediatedtransfection, viral vectors and the transfection methods described byLife Technologies Ltd, Paisley, UK using Lipofectamin 2000 and RocheDiagnostics Corporation, Indianapolis, USA using FuGENE 6. These methodsare well known in the art and are described by Ausbel et al. (eds.),1996, Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, USA. The cultivation of mammalian cells may be performed accordingto established methods, e.g. as disclosed in (Animal Cell Biotechnology,Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc.Totowa, N.J., USA and Harrison Mass. and Rae I F, General Techniques ofCell Culture, Cambridge University Press 1997).

Expression Systems, Culture, and Isolation

Insulin polypeptides may be expressed in any number of suitableexpression systems including, for example, yeast, insect cells,mammalian cells, and bacteria. A description of exemplary expressionsystems is provided below.

Yeast As used herein, the term “yeast” includes any of the variousyeasts capable of expressing a gene encoding an insulin polypeptide.Such yeasts include, but are not limited to, ascosporogenous yeasts(Endomycetales), basidiosporogenous yeasts and yeasts belonging to theFungi imperfecti (Blastomycetes) group. The ascosporogenous yeasts aredivided into two families, Spermophthoraceae and Saccharomycetaceae. Thelatter is comprised of four subfamilies, Sehizosaccharomycoideae (e.g.,genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeasts belonging to the Fungi Imperfecti (Blastomycetes)group are divided into two families, Sporobolomycetaceae (e.g., generaSporobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida).

Of particular interest for use with the present invention are specieswithin the genera Pichia, Kluyveromyces, Saccharomyces,Schizosaccharomyces, Hansenula, Torulopsis, and Candida, including, butnot limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S.carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis,S. oviformis, K. lactis, K. fragilis, C. albicans, C. maltosa, and H.polymorpha.

The selection of suitable yeast for expression of insulin polypeptidesis within the skill of one of ordinary skill in the art. In selectingyeast hosts for expression, suitable hosts may include those shown tohave, for example, good secretion capacity, low proteolytic activity,good secretion capacity, good soluble protein production, and overallrobustness. Yeast are generally available from a variety of sourcesincluding, but not limited to, the Yeast Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.), and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

The term “yeast host” or “yeast host cell” includes yeast that can be,or has been, used as a recipient for recombinant vectors or othertransfer DNA. The term includes the progeny of the original yeast hostcell that has received the recombinant vectors or other transfer DNA. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding a insulin polypeptide, areincluded in the progeny intended by this definition.

Expression and transformation vectors, including extrachromosomalreplicons or integrating vectors, have been developed for transformationinto many yeast hosts. For example, expression vectors have beendeveloped for S. cerevisiae (Sikorski et al., GENETICS (1989) 122:19;Ito et al., J. BACTERIOL. (1983) 153:163; Hinnen et al., PROC. NATL.ACAD. SCI. USA (1978) 75:1929); C. albicans (Kurtz et al., MOL. CELL.BIOL. (1986) 6:142); C. maltosa (Kunze et al., J. BASIC MICROBIOL.(1985) 25:141); H. polymorpha (Gleeson et al., J. GEN. MICROBIOL. (1986)132:3459; Roggenkamp et al., MOL. GENETICS AND GENOMICS (1986) 202:302);K. fragilis (Das et al., J. BACTERIOL. (1984) 158:1165); K. lactis (DeLouvencourt et al., J. BACTERIOL. (1983) 154:737; Van den Berg et al.,BIOTECHNOLOGY (NY) (1990) 8:135); P. guillerimondii (Kunze et al., J.BASIC MICROBIOL. (1985) 25:141); P. pastoris (U.S. Pat. Nos. 5,324,639;4,929,555; and 4,837,148; Cregg et al., MOL. CELL. BIOL. (1985) 5:3376);Schizosaccharomyces pombe (Beach et al., NATURE (1982) 300:706); and Y.lipolytica; A. nidulans (Ballance et al., BIOCHEM. BIOPHYS. RES. COMMUN.(1983) 112:284-89; Tilburn et al., GENE (1983) 26:205-221; and Yelton etal., PROC. NATL. ACAD. SCI. USA (1984) 81:1470-74); A. niger (Kelly andHynes, EMBO J. (1985) 4:475-479); T. recesia (EP 0 244 234); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357), each incorporated by reference herein.

Control sequences for yeast vectors are known to those of ordinary skillin the art and include, but are not limited to, promoter regions fromgenes such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolase;glucokinase; glucose-6-phosphate isomerase;glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH); hexokinase;phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase(PyK) (EP 0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also may provide useful promoter sequences (Miyanohara et al., PROC.NATL. ACAD. SCI. USA (1983) 80:1). Other suitable promoter sequences foruse with yeast hosts may include the promoters for 3-phosphoglyceratekinase (Hitzeman et al., J. BIOL. CHEM. (1980) 255:12073); and otherglycolytic enzymes, such as pyruvate decarboxylase, triosephosphateisomerase, and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY(1978) 17:4900; Hess et al., J. ADV. ENZYME REG. (1969) 7:149).Inducible yeast promoters having the additional advantage oftranscription controlled by growth conditions may include the promoterregions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase;metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degradativeenzymes associated with nitrogen metabolism; and enzymes responsible formaltose and galactose utilization. Suitable vectors and promoters foruse in yeast expression are further described in EP 0 073 657.

Yeast enhancers also may be used with yeast promoters. In addition,synthetic promoters may also function as yeast promoters. For example,the upstream activating sequences (UAS) of a yeast promoter may bejoined with the transcription activation region of another yeastpromoter, creating a synthetic hybrid promoter. Examples of such hybridpromoters include the ADH regulatory sequence linked to the GAPtranscription activation region. See U.S. Pat. Nos. 4,880,734 and4,876,197, which are incorporated by reference herein. Other examples ofhybrid promoters include promoters that consist of the regulatorysequences of the ADH2, GAL4, GAL10, or PHO5 genes, combined with thetranscriptional activation region of a glycolytic enzyme gene such asGAP or PyK. See EP 0 164 556. Furthermore, a yeast promoter may includenaturally occurring promoters of non-yeast origin that have the abilityto bind yeast RNA polymerase and initiate transcription.

Other control elements that may comprise part of the yeast expressionvectors include terminators, for example, from GAPDH or the enolasegenes (Holland et al., 0.1. BIOL. CHEM. (1981) 256:1385). In addition,the origin of replication from the 2μ plasmid origin is suitable foryeast. A suitable selection gene for use in yeast is the trp1 genepresent in the yeast plasmid. See Tschumper et al., GENE (1980) 10:157;Kingsman et al., GENE (1979) 7:141. The trp1 gene provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Methods of introducing exogenous DNA into yeast hosts are known to thoseof ordinary skill in the art, and typically include, but are not limitedto, either the transformation of spheroplasts or of intact yeast hostcells treated with alkali cations. For example, transformation of yeastcan be carried out according to the method described in Hsiao et al.,PROC. NATL. ACAD. SCI. USA (1979) 76:3829 and Van Solingen et al., J.BACT. (1977) 130:946. However, other methods for introducing DNA intocells such as by nuclear injection, electroporation, or protoplastfusion may also be used as described generally in SAMBROOK ET AL.,MOLECULAR CLONING: A LAB. MANUAL (2001). Yeast host cells may then becultured using standard techniques known to those of ordinary skill inthe art.

Other methods for expressing heterologous proteins in yeast host cellsare known to those of ordinary skill in the art. See generally U.S.Patent Publication No. 20020055169, U.S. Pat. Nos. 6,361,969; 6,312,923;6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and5,089,398; U.S. Reexamined Patent Nos. RE37,343 and RE35,749; PCTPublished Patent Applications WO 99/07862; WO 98/37208; and WO 98/26080;European Patent Applications EP 0 946 736; EP 0 732 403; EP 0 480 480;WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556.See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62(1-2):79-93;Romanos et al., YEAST (1992) 8(6):423-488; Goeddel, METHODS INENZYMOLOGY (1990) 185:3-7, each incorporated by reference herein.

The yeast host strains may be grown in fermentors during theamplification stage using standard feed batch fermentation methods knownto those of ordinary skill in the art. The fermentation methods may beadapted to account for differences in a particular yeast host's carbonutilization pathway or mode of expression control. For example,fermentation of a Saccharomyces yeast host may require a single glucosefeed, complex nitrogen source (e.g., casein hydrolysates), and multiplevitamin supplementation. In contrast, the methylotrophic yeast P.pastoris may require glycerol, methanol, and trace mineral feeds, butonly simple ammonium (nitrogen) salts for optimal growth and expression.See, e.g., U.S. Pat. No. 5,324,639; Elliott et al., J. PROTEIN CHEM.(1990) 9:95; and Fieschko et al., BIOTECH. BIOENG. (1987) 29:1113,incorporated by reference herein.

Such fermentation methods, however, may have certain common featuresindependent of the yeast host strain employed. For example, a growthlimiting nutrient, typically carbon, may be added to the fermentorduring the amplification phase to allow maximal growth. In addition,fermentation methods generally employ a fermentation medium designed tocontain adequate amounts of carbon, nitrogen, basal salts, phosphorus,and other minor nutrients (vitamins, trace minerals and salts, etc.).Examples of fermentation media suitable for use with Pichia aredescribed in U.S. Pat. Nos. 5,324,639 and 5,231,178, which areincorporated by reference herein.

Baculovirus-Infected Insect Cells The term “insect host” or “insect hostcell” refers to a insect that can be, or has been, used as a recipientfor recombinant vectors or other transfer DNA. The term includes theprogeny of the original insect host cell that has been transfected. Itis understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement to the original parent, due to accidental or deliberatemutation. Progeny of the parental cell that are sufficiently similar tothe parent to be characterized by the relevant property, such as thepresence of a nucleotide sequence encoding an insulin polypeptide, areincluded in the progeny intended by this definition. Baculovirusexpression of insulin polypeptides is useful in the present inventionand the use of rDNA technology, polypeptides or precursors thereofbecause insulin may be biosynthesized in any number of host cellsincluding bacteria, mammalian cells, insect cells, yeast or fungi. Anembodiment of the present invention includes biosynthesis of insulin,modified insulin, insulin polypeptides, or insulin analogs in bacteria,yeast or mammalian cells. Another embodiment of the present inventioninvolves biosynthesis done in E. coli or a yeast. Examples ofbiosynthesis in mammalian cells and transgenic animals are described inHakola, K. [Molecular and Cellular Endocrinology, 127:59-69, (1997)].

The selection of suitable insect cells for expression of insulinpolypeptides is known to those of ordinary skill in the art. Severalinsect species are well described in the art and are commerciallyavailable including Aedes aegypti, Bombyx mori, Drosophila melanogaster,Spodoptera frugiperda, and Trichoplusia ni. In selecting insect hostsfor expression, suitable hosts may include those shown to have, interglia, good secretion capacity, low proteolytic activity, and overallrobustness. Insect are generally available from a variety of sourcesincluding, but not limited to, the Insect Genetic Stock Center,Department of Biophysics and Medical Physics, University of California(Berkeley, Calif.); and the American Type Culture Collection (“ATCC”)(Manassas, Va.).

Generally, the components of a baculovirus-infected insect expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene to be expressed;a wild type baculovirus with sequences homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.The materials, methods and techniques used in constructing vectors,transfecting cells, picking plaques, growing cells in culture, and thelike are known in the art and manuals are available describing thesetechniques.

After inserting the heterologous gene into the transfer vector, thevector and the wild type viral genome are transfected into an insecthost cell where the vector and viral genome recombine. The packagedrecombinant virus is expressed and recombinant plaques are identifiedand purified. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, forexample, Invitrogen Corp. (Carlsbad, Calif.). These techniques aregenerally known to those of ordinary skill in the art and fullydescribed in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATIONBULLETIN NO. 1555 (1987), herein incorporated by reference. See also,RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSIONPROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORYGUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992).

Indeed, the production of various heterologous proteins usingbaculovirus/insect cell expression systems is known to those of ordinaryskill in the art. See, e.g., U.S. Pat. Nos. 6,368,825; 6,342,216;6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932;6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676;5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220;5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032; WO99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO89/01037; WO 88/07082, which are incorporated by reference herein.

Vectors that are useful in baculovirus/insect cell expression systemsare known in the art and include, for example, insect expression andtransfer vectors derived from the baculovirus Autographacalifornicanuclear polyhedrosis virus (AcNPV), which is a helper-independent, viralexpression vector. Viral expression vectors derived from this systemusually use the strong viral polyhedrin gene promoter to driveexpression of heterologous genes. See generally, O'Reilly ET AL.,BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, theabove-described components, comprising a promoter, leader (if desired),coding sequence of interest, and transcription termination sequence, aretypically assembled into an intermediate transplacement construct(transfer vector). Intermediate transplacement constructs are oftenmaintained in a replicon, such as an extra chromosomal element (e.g.,plasmids) capable of stable maintenance in a host, such as bacteria. Thereplicon will have a replication system, thus allowing it to bemaintained in a suitable host for cloning and amplification. Morespecifically, the plasmid may contain the polyhedrin polyadenylationsignal (Miller, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

One commonly used transfer vector for introducing foreign genes intoAcNPV is pAc373. Many other vectors, known to those of skill in the art,have also been designed including, for example, pVL985, which alters thepolyhedrin start codon from ATG to ATT, and which introduces a BamHIcloning site 32 base pairs downstream from the ATT, See Luckow andSummers, VIROLOGY 170:31 (1989). Other commercially available vectorsinclude, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac;pBlueBac4.5 (Invitrogen Corp., Carlsbad, Calif.).

After insertion of the heterologous gene, the transfer vector and wildtype baculoviral genome are co-transfected into an insect cell host.Methods for introducing heterologous DNA into the desired site in thebaculovirus virus are known in the art, See SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al.,MOL. CELL. BIOL. (1983) 3:2156; Luckow and Summers, VIROLOGY (1989)170:31. For example, the insertion can be into a gene such as thepolyhedrin gene, by homologous double crossover recombination; insertioncan also be into a restriction enzyme site engineered into the desiredbaculovirus gene. See Miller et al., BIOESSAYS (1989) 11(4):91.

Transfection may be accomplished by electroporation. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN.VIROL. (1989) 70:3501. Alternatively, liposomes may be used to transfectthe insect cells with the recombinant expression vector and thebaculovirus. See, e.g., Liebman et al., BIOTECHNIQUES (1999) 26(1):36;Graves et al., BIOCHEMISTRY (1998) 37:6050; Nomura et al., J. BIOL.CHEM. (1998) 273(22):13570; Schmidt et al., PROTEIN EXPRESSION ANDPURIFICATION (1998) 12:323; Siffert et al., NATURE GENETICS (1998)18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154(1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263;Dolphin et al., NATURE GENETICS (1997) 17:491; Kost et al., GENE (1997)190:139; Jakobsson et al., J. BIOL. CHEM. (1996) 271:22203; Rowles etal., J. BIOL. CHEM. (1996) 271(37):22376; Reverey et al., J. BIOL. CHEM.(1996) 271(39):23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270:4121;Sisk et al., J. VIROL. (1994) 68(2):766; and Peng et al., BIOTECHNIQUES(1993) 14(2):274. Commercially available liposomes include, for example,Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, Calif.). Inaddition, calcium phosphate transfection may be used. See TROTTER ANDWOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990)18(19):5667; and Mann and King, J. GEN. VIROL. (1989) 70:3501.

Baculovirus expression vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (3′)transcription of a coding sequence (e.g., structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region typically includes an RNA polymerase binding site anda transcription initiation site. A baculovirus promoter may also have asecond domain called an enhancer, which, if present, is usually distalto the structural gene. Moreover, expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in the infectioncycle, provide particularly useful promoter sequences. Examples includesequences derived from the gene encoding the viral polyhedron protein(FRIESEN ET AL., The Regulation of Baculovirus Gene Expression in THEMOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476)and the gene encoding the p10 protein (Vlak et al., J. GEN. VIROL.(1988) 69:765).

The newly formed baculovirus expression vector is packaged into aninfectious recombinant baculovirus and subsequently grown plaques may bepurified by techniques known to those of ordinary skill in the art. SeeMiller et al., BIOESSAYS (1989) 11(4):91; SUMMERS AND SMITH, TEXASAGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia, Aedes aegypti (ATCCNo. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophila melanogaster(ATCC No. 1963), Spodoptera frugiperda, and Trichoplusia ni. See Wright,NATURE (1986) 321:718; Carbonell et al., J. VIROL. (1985) 56:153; Smithet al., MOL. CELL. BIOL. (1983) 3:2156. See generally, Fraser et al., INVITRO CELL. DEV. BIOL. (1989) 25:225. More specifically, the cell linesused for baculovirus expression vector systems commonly include, but arenot limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21(Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad,Calif.)), Tri-368 (Trichopulsia ni), and High-Five™ BTI-TN-5B1-4(Trichopulsia ni).

Cells and culture media are commercially available for both direct andfusion expression of heterologous polypeptides in abaculovirus/expression, and cell culture technology is generally knownto those of ordinary skill in the art.

E. Coli., Pseudomonas species, and other Prokaryotes Bacterialexpression techniques are known to those of ordinary skill in the art. Awide variety of vectors are available for use in bacterial hosts. Thevectors may be single copy or low or high multicopy vectors. Vectors mayserve for cloning and/or expression. In view of the ample literatureconcerning vectors, commercial availability of many vectors, and evenmanuals describing vectors and their restriction maps andcharacteristics, no extensive discussion is required here. As iswell-known, the vectors normally involve markers allowing for selection,which markers may provide for cytotoxic agent resistance, prototrophy orimmunity. Frequently, a plurality of markers is present, which providefor different characteristics.

A bacterial promoter is any DNA sequence capable of binding bacterialRNA polymerase and initiating the downstream (3′) transcription of acoding sequence (e.g. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regiontypically includes an RNA polymerase binding site and a transcriptioninitiation site. A bacterial promoter may also have a second domaincalled an operator, that may overlap an adjacent RNA polymerase bindingsite at which RNA synthesis begins. The operator permits negativeregulated (inducible) transcription, as a gene repressor protein maybind the operator and thereby inhibit transcription of a specific gene.Constitutive expression may occur in the absence of negative regulatoryelements, such as the operator. In addition, positive regulation may beachieved by a gene activator protein binding sequence, which, if presentis usually proximal (5′) to the RNA polymerase binding sequence, Anexample of a gene activator protein is the catabolite activator protein(CAP), which helps initiate transcription of the lac operon inEscherichia coli (E. coli) [Raibaud et al., ANNU. REV. GENET. (1984)18:173]. Regulated expression may therefore be either positive ornegative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal., NATURE (1977) 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8:4057; Yelverton et al.,NUCL. ACIDS RES. (1981) 9:731; U.S. Pat. No. 4,738,921; EP Pub. Nos. 036776 and 121 775, which are incorporated by reference herein]. Theβ-galactosidase (bla) promoter system [Weissmann (1981) “The cloning ofinterferon and other mistakes.” In Interferon 3 (Ed. I. Gresser)],bacteriophage lambda PL [Shimatake et al., NATURE (1981) 292:128] and T5[U.S. Pat. No. 4,689,406, which are incorporated by reference herein]promoter systems also provide useful promoter sequences. Preferredmethods of the present invention utilize strong promoters, such as theT7 promoter to induce insulin polypeptides at high levels. Examples ofsuch vectors are known to those of ordinary skill in the art and includethe pET29 series from Novagen, and the pPOP vectors described inWO99/05297, which is incorporated by reference herein. Such expressionsystems produce high levels of insulin polypeptides in the host withoutcompromising host cell viability or growth parameters. pET19 (Novagen)is another vector known in the art.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters, For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433, which isincorporated by reference herein]. For example, the tac promoter is ahybrid trp-lac promoter comprised of both trp promoter and lac operonsequences that is regulated by the lac repressor [Amann et al., GENE(1983) 25:167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21].Furthermore, a bacterial promoter can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al., J.MOL. BIOL. (1986) 189:113; Tabor et al., Proc Natl. Acad. Sci. (1985)82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EP Pub. No. 267851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al., NATURE (1975) 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. “Genetic signals and nucleotide sequences inmessenger RNA”, In Biological Regulation and Development: GeneExpression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genesand prokaryotic genes with weak ribosome-binding site [Sambrook et al.“Expression of cloned genes in Escherichia coli”, Molecular Cloning: ALaboratory Manual, 1989].

The term “bacterial host” or “bacterial host cell” refers to a bacterialthat can be, or has been, used as a recipient for recombinant vectors orother transfer DNA. The term includes the progeny of the originalbacterial host cell that has been transfected. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement to theoriginal parent, due to accidental or deliberate mutation. Progeny ofthe parental cell that are sufficiently similar to the parent to becharacterized by the relevant property, such as the presence of anucleotide sequence encoding an insulin polypeptide, are included in theprogeny intended by this definition.

The selection of suitable host bacteria for expression of insulinpolypeptides is known to those of ordinary skill in the art. Inselecting bacterial hosts for expression, suitable hosts may includethose shown to have, inter alia, good inclusion body formation capacity,low proteolytic activity, and overall robustness. Bacterial hosts aregenerally available from a variety of sources including, but not limitedto, the Bacterial Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, Calif.); and theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.).Industrial/pharmaceutical fermentation generally use bacterial derivedfrom K strains (e.g. W3110) or from bacteria derived from B strains(e.g. BL21). These strains are particularly useful because their growthparameters are extremely well known and robust. In addition, thesestrains are non-pathogenic, which is commercially important for safetyand environmental reasons. Other examples of suitable E. coli hostsinclude, but are not limited to, strains of BL21, DH10B, or derivativesthereof. In another embodiment of the methods of the present invention,the E. coli host is a protease minus strain including, but not limitedto, OMP- and LON-. The host cell strain may be a species of Pseudomonas,including but not limited to, Pseudomonas fluorescens, Pseudomonasaeruginosa, and Pseudomonas putida. Pseudomonas fluorescens biovar 1,designated strain MB101, is known to be useful for recombinantproduction and is available for therapeutic protein productionprocesses. Examples of a Pseudomonas expression system include thesystem available from The Dow Chemical Company as a host strain(Midland, Mich. available on the World Wide Web at dow.com).

Once a recombinant host cell strain has been established (i.e., theexpression construct has been introduced into the host cell and hostcells with the proper expression construct are isolated), therecombinant host cell strain is cultured under conditions appropriatefor production of insulin polypeptides. As will be apparent to one ofskill in the art, the method of culture of the recombinant host cellstrain will be dependent on the nature of the expression constructutilized and the identity of the host cell. Recombinant host strains arenormally cultured using methods that are known to those of ordinaryskill in the art. Recombinant host cells are typically cultured inliquid medium containing assimilatable sources of carbon, nitrogen, andinorganic salts and, optionally, containing vitamins, amino acids,growth factors, and other proteinaceous culture supplements known tothose of ordinary skill in the art. Liquid media for culture of hostcells may optionally contain antibiotics or anti-fungals to prevent thegrowth of undesirable microorganisms and/or compounds including, but notlimited to, antibiotics to select for host cells containing theexpression vector.

Recombinant host cells may be cultured in batch or continuous formats,with either cell harvesting (in the case where the insulin polypeptideaccumulates intracellularly) or harvesting of culture supernatant ineither batch or continuous formats. For production in prokaryotic hostcells, batch culture and cell harvest are preferred.

The insulin polypeptides of the present invention are normally purifiedafter expression in recombinant systems. The insulin polypeptide may bepurified from host cells or culture medium by a variety of methods knownto the art. Insulin polypeptides produced in bacterial host cells may bepoorly soluble or insoluble (in the form of inclusion bodies). In oneembodiment of the present invention, amino acid substitutions mayreadily be made in the insulin polypeptide that are selected for thepurpose of increasing the solubility of the recombinantly producedprotein utilizing the methods disclosed herein as well as those known inthe art. In the case of insoluble protein, the protein may be collectedfrom host cell lysates by centrifugation and may further be followed byhomogenization of the cells. In the case of poorly soluble protein,compounds including, but not limited to, polyethylene imine (PEI) may beadded to induce the precipitation of partially soluble protein. Theprecipitated protein may then be conveniently collected bycentrifugation. Recombinant host cells may be disrupted or homogenizedto release the inclusion bodies from within the cells using a variety ofmethods known to those of ordinary skill in the art. Host celldisruption or homogenization may be performed using well knowntechniques including, but not limited to, enzymatic cell disruption,sonication, dounce homogenization, or high pressure release disruption.In one embodiment of the method of the present invention, the highpressure release technique is used to disrupt the E. coli host cells torelease the inclusion bodies of the insulin polypeptides. When handlinginclusion bodies of insulin polypeptide, it may be advantageous tominimize the homogenization time on repetitions in order to maximize theyield of inclusion bodies without loss due to factors such assolubilization, mechanical shearing or proteolysis.

Insoluble or precipitated insulin polypeptide may then be solubilizedusing any of a number of suitable solubilization agents known to theart. The insulin polyeptide may be solubilized with urea or guanidinehydrochloride. The volume of the solubilized insulin polypeptide shouldbe minimized so that large batches may be produced using convenientlymanageable batch sizes. This factor may be significant in a large-scalecommercial setting where the recombinant host may be grown in batchesthat are thousands of liters in volume. In addition, when manufacturinginsulin polypeptide in a large-scale commercial setting, in particularfor human pharmaceutical uses, the avoidance of harsh chemicals that candamage the machinery and container, or the protein product itself,should be avoided, if possible. It has been shown in the method of thepresent invention that the milder denaturing agent urea can be used tosolubilize the insulin polypeptide inclusion bodies in place of theharsher denaturing agent guanidine hydrochloride. The use of ureasignificantly reduces the risk of damage to stainless steel equipmentutilized in the manufacturing and purification process of insulinpolypeptide while efficiently solubilizing the insulin polypeptideinclusion bodies.

In the case of soluble insulin protein, the insulin may be secreted intothe periplasmic space or into the culture medium. In addition, solubleinsulin may be present in the cytoplasm of the host cells. It may bedesired to concentrate soluble insulin prior to performing purificationsteps. Standard techniques known to those of ordinary skill in the artmay be used to concentrate soluble insulin from, for example, celllysates or culture medium. In addition, standard techniques known tothose of ordinary skill in the art may be used to disrupt host cells andrelease soluble insulin from the cytoplasm or periplasmic space of thehost cells.

When insulin polypeptide is produced as a fusion protein, the fusionsequence may be removed. Removal of a fusion sequence may beaccomplished by enzymatic or chemical cleavage. Enzymatic removal offusion sequences may be accomplished using methods known to those ofordinary skill in the art. The choice of enzyme for removal of thefusion sequence will be determined by the identity of the fusion, andthe reaction conditions will be specified by the choice of enzyme aswill be apparent to one of ordinary skill in the art. Chemical cleavagemay be accomplished using reagents known to those of ordinary skill inthe art, including but not limited to, cyanogen bromide, TEV protease,and other reagents. The cleaved insulin polypeptide may be purified fromthe cleaved fusion sequence by methods known to those of ordinary skillin the art. Such methods will be determined by the identity andproperties of the fusion sequence and the insulin polypeptide, as willbe apparent to one of ordinary skill in the art. Methods forpurification may include, but are not limited to, size-exclusionchromatography, hydrophobic interaction chromatography, ion-exchangechromatography or dialysis or any combination thereof.

The insulin polypeptide may also be purified to remove DNA from theprotein solution. DNA may be removed by any suitable method known to theart, such as precipitation or ion exchange chromatography, but may beremoved by precipitation with a nucleic acid precipitating agent, suchas, but not limited to, protamine sulfate. The insulin polypeptide maybe separated from the precipitated DNA using standard well known methodsincluding, but not limited to, centrifugation or filtration. Removal ofhost nucleic acid molecules is an important factor in a setting wherethe insulin polypeptide is to be used to treat humans and the methods ofthe present invention reduce host cell DNA to pharmaceuticallyacceptable levels.

Methods for small-scale or large-scale fermentation can also be used inprotein expression, including but not limited to, fermentors, shakeflasks, fluidized bed bioreactors, hollow fiber bioreactors, rollerbottle culture systems, and stirred tank bioreactor systems. Each ofthese methods can be performed in a batch, fed-batch, or continuous modeprocess.

Human insulin polypeptides of the invention can generally be recoveredusing methods standard in the art. For example, culture medium or celllysate can be centrifuged or filtered to remove cellular debris. Thesupernatant may be concentrated or diluted to a desired volume ordiafiltered into a suitable buffer to condition the preparation forfurther purification. Further purification of the insulin polypeptide ofthe present invention includes separating deamidated and clipped formsof the insulin polypeptide variant from the intact form.

Any of the following exemplary procedures can be employed forpurification of insulin polypeptides of the invention: affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;high performance liquid chromatography (HPLC); reverse phase HPLC; gelfiltration (using, including but not limited to, SEPHADEX G-75);hydrophobic interaction chromatography; size-exclusion chromatography;metal-chelate chromatography; ultrafiltration/diafiltration; ethanolprecipitation; ammonium sulfate precipitation; chromatofocusing;displacement chromatography; electrophoretic procedures (including butnot limited to preparative isoelectric focusing), differentialsolubility (including but not limited to ammonium sulfateprecipitation), SDS-PAGE, or extraction.

Proteins of the present invention, including but not limited to,proteins comprising unnatural amino acids, peptides comprising unnaturalamino acids, antibodies to proteins comprising unnatural amino acids,binding partners for proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods known to those of ordinary skillin the art, including but not limited to, ammonium sulfate or ethanolprecipitation, acid or base extraction, column chromatography, affinitycolumn chromatography, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,hydroxylapatite chromatography, lectin chromatography, gelelectrophoresis and the like. Protein refolding steps can be used, asdesired, in making correctly folded mature proteins. High performanceliquid chromatography (HPLC), affinity chromatography or other suitablemethods can be employed in final purification steps where high purity isdesired. In one embodiment, antibodies made against unnatural aminoacids (or proteins or peptides comprising unnatural amino acids) areused as purification reagents, including but not limited to, foraffinity-based purification of proteins or peptides comprising one ormore unnatural amino acid(s). Once purified, partially or tohomogeneity, as desired, the polypeptides are optionally used for a widevariety of utilities, including but not limited to, as assay components,therapeutics, prophylaxis, diagnostics, research reagents, and/or asimmunogens for antibody production. Antibodies generated againstpolypeptides of the present invention may be obtained by administeringthe polypeptides or epitope-bearing fragments, or cells to an animal,preferably a non-human animal, using routine protocols. One of ordinaryskill in the art could generate antibodies using a variety of knowntechniques. Also, transgenic mice, or other organisms, including othermammals, may be used to express humanized antibodies. Theabove-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or to purify the polypeptides.Antibodies against polypeptides of the present invention may also beemployed to treat diseases.

Polypeptides and polynucleotides of the present invention may also beused as vaccines. Accordingly, in a further aspect, the presentinvention relates to a method for inducing an immunological response ina mammal that comprises inoculating the mammal with a polypeptide of thepresent invention, adequate to produce antibody and/or T cell immuneresponse, including, for example, cytokine-producing T cells orcytotoxic T cells, to protect said animal from disease, whether thatdisease is already established within the individual or not. Animmunological response in a mammal may also be induced by a methodcomprises delivering a polypeptide of the present invention via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect said animal from diseases of the invention.One way of administering the vector is by accelerating it into thedesired cells as a coating on particles or otherwise. Such nucleic acidvector may comprise DNA, RNA, a modified nucleic acid, or a DNA/RNAhybrid. For use as a vaccine, a polypeptide or a nucleic acid vectorwill be normally provided as a vaccine formulation (composition). Theformulation may further comprise a suitable carrier. Since a polypeptidemay be broken down in the stomach, it may be administered parenterally(for instance, subcutaneous, intramuscular, intravenous, or intra-dermalinjection). Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions that may containanti-oxidants, buffers, bacteriostats and solutes that render theformulation instonic with the blood of the recipient; and aqueous andnon-aqueous sterile suspensions that may include suspending agents orthickening agents. The vaccine formulation may also include adjuvantsystems for enhancing the immunogenicity of the formulation which areknown to those of ordinary skill in the art. The dosage will depend onthe specific activity of the vaccine and can be readily determined byroutine experimentation.

Expression in Alternate Systems

Several strategies have been employed to introduce unnatural amino acidsinto proteins in non-recombinant host cells, mutagenized host cells, orin cell-free systems. These systems are also suitable for use in makingthe Insulin polypeptides of the present invention. Derivatization ofamino acids with reactive side-chains such as Lys, Cys and Tyr resultedin the conversion of lysine to N²-acetyl-lysine. Chemical synthesis alsoprovides a straightforward method to incorporate unnatural amino acids.With the recent development of enzymatic ligation and native chemicalligation of peptide fragments, it is possible to make larger proteins.See, e.g., P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem, 69:923(2000). Chemical peptide ligation and native chemical ligation aredescribed in. U.S. Pat. No. 6,184,344, U.S. Patent Publication No.2004/0138412, U.S. Patent Publication No. 2003/0208046, WO 02/098902,and WO 03/042235, which are incorporated by reference herein. A generalin vitro biosynthetic method in which a suppressor tRNA chemicallyacylated with the desired unnatural amino acid is added to an in vitroextract capable of supporting protein biosynthesis, has been used tosite-specifically incorporate over 100 unnatural amino acids into avariety of proteins of virtually any size. See, e.g., V. W. Cornish, D.Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621(1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244:182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111:8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction.

In addition to other references noted herein, a variety ofpurification/protein folding methods are known to those of ordinaryskill in the art, including, but not limited to, those set forth in R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification, AcademicPress, Inc. N.Y. (1990); Sandana, (1997) Bioseparation of Proteins,Academic Press, Inc.; Bollag et al. (1996) Protein Methods, 2nd EditionWiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook. HumanaPress, NJ, Harris and Angal, (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal, Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes, (1993) Protein Purification: Principlesand Practice 3rd Edition Springer Verlag, NY; Janson and Ryden, (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; and the references cited therein.

One advantage of producing a protein or polypeptide of interest with anunnatural amino acid in a eukaryotic host cell or non-eukaryotic hostcell is that typically the proteins or polypeptides will be folded intheir native conformations. However, in certain embodiments of theinvention, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins or peptides canpossess a conformation different from the desired conformations of therelevant polypeptides. In one aspect of the invention, the expressedprotein or polypeptide is optionally denatured and then renatured. Thisis accomplished utilizing methods known in the art, including but notlimited to, by adding a chaperonin to the protein or polypeptide ofinterest, by solubilizing the proteins in a chaotropic agent such asguanidine HCl, utilizing protein disulfide isomerase, etc.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are known tothose of ordinary skill in the art (see, the references above, andDebinski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman andPastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992)Anal. Biochem., 205: 263-270). Debinski, et al., for example, describethe denaturation and reduction of inclusion body proteins inguanidine-DTE. The proteins can be refolded in a redox buffercontaining, including but not limited to, oxidized glutathione andL-arginine. Refolding reagents can be flowed or otherwise moved intocontact with the one or more polypeptide or other expression product, orvice-versa.

In the case of prokaryotic production of insulin polypeptide, theinsulin polypeptide thus produced may be misfolded and thus lacks or hasreduced biological activity. The bioactivity of the protein may berestored by “refolding”. In general, misfolded insulin polypeptide isrefolded by solubilizing (where the insulin polypeptide is alsoinsoluble), unfolding and reducing the polypeptide chain using, forexample, one or more chaotropic agents (e.g. urea and/or guanidine) anda reducing agent capable of reducing disulfide bonds (e.g.dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At a moderateconcentration of chaotrope, an oxidizing agent is then added (e.g.,oxygen, cystine or cystamine), which allows the reformation of disulfidebonds. Insulin polypeptide may be refolded using standard methods knownin the art, such as those described in U.S. Pat. Nos. 4,511,502,4,511,503, and 4,512,922, which are incorporated by reference herein.The insulin polypeptide may also be cofolded with other proteins to formheterodimers or heteromultimers.

After refolding, the insulin may be further purified. Purification ofinsulin may be accomplished using a variety of techniques known to thoseof ordinary skill in the art, including hydrophobic interactionchromatography, size exclusion chromatography, ion exchangechromatography, reverse-phase high performance liquid chromatography,affinity chromatography, and the like or any combination thereof.Additional purification may also include a step of drying orprecipitation of the purified protein.

After purification, insulin may be exchanged into different buffersand/or concentrated by any of a variety of methods known to the art,including, but not limited to, diafiltration and dialysis. Insulin thatis provided as a single purified protein may be subject to aggregationand precipitation.

The purified insulin may be at least 90% pure (as measured by reversephase high performance liquid chromatography, RP-HPLC, or sodium dodecylsulfate-polyacrylamide gel electrophoresis, SDS-PAGE) or at least 95%pure, or at least 98% pure, or at least 99% or greater pure. Regardlessof the exact numerical value of the purity of the insulin, the insulinis sufficiently pure for use as a pharmaceutical product or for furtherprocessing, such as conjugation with a water soluble polymer such asPEG.

Certain insulin molecules may be used as therapeutic agents in theabsence of other active ingredients or proteins (other than excipients,carriers, and stabilizers, serum albumin and the like), or they may becomplexed with another protein or a polymer.

General Purification Methods Any one of a variety of isolation steps maybe performed on the cell lysate, extract, culture medium, inclusionbodies, periplasmic space of the host cells, cytoplasm of the hostcells, or other material, comprising insulin polypeptide or on anyinsulin polypeptide mixtures resulting from any isolation stepsincluding, but not limited to, affinity chromatography, ion exchangechromatography, hydrophobic interaction chromatography, gel filtrationchromatography, high performance liquid chromatography (“HPLC”),reversed phase-HPLC (“RP-HPLC”), expanded bed adsorption, or anycombination and/or repetition thereof and in any appropriate order.

Equipment and other necessary materials used in performing thetechniques described herein are commercially available. Pumps, fractioncollectors, monitors, recorders, and entire systems are available from,for example, Applied Biosystems (Foster City, Calif.), Bio-RadLaboratories, Inc. (Hercules, Calif.), and Amersham Biosciences, Inc.(Piscataway, N.J.). Chromatographic materials including, but not limitedto, exchange matrix materials, media, and buffers are also availablefrom such companies.

Equilibration, and other steps in the column chromatography processesdescribed herein such as washing and elution, may be more rapidlyaccomplished using specialized equipment such as a pump. Commerciallyavailable pumps include, but are not limited to, HILOAD® Pump P-50,Peristaltic Pump P-1, Pump P-901, and Pump P-903 (Amersham Biosciences,Piscataway, N.J.).

Examples of fraction collectors include RediFrac Fraction Collector,FRAC-100 and FRAC-200 Fraction Collectors, and SUPERFRAC® FractionCollector (Amersham Biosciences, Piscataway, N.J.). Mixers are alsoavailable to form pH and linear concentration gradients. Commerciallyavailable mixers include Gradient Mixer GM-1 and In-Line Mixers(Amersham Biosciences, Piscataway, N.J.).

The chromatographic process may be monitored using any commerciallyavailable monitor. Such monitors may be used to gather information likeUV, pH, and conductivity. Examples of detectors include Monitor UV-1,UVICORD® S II, Monitor UV-M II, Monitor UV-900, Monitor UPC-900, MonitorpH/C-900, and Conductivity Monitor (Amersham Biosciences, Piscataway,N.J.). Indeed, entire systems are commercially available including thevarious AKTA® systems from Amersham Biosciences (Piscataway, N.J.).

In one embodiment of the present invention, for example, the insulinpolypeptide may be reduced and denatured by first denaturing theresultant purified insulin polypeptide in urea, followed by dilutioninto TRIS buffer containing a reducing agent (such as DTT) at a suitablepH. In another embodiment, the insulin polypeptide is denatured in ureain a concentration range of between about 2 M to about 9 M, followed bydilution in TRIS buffer at a pH in the range of about 5.0 to about 8.0.The refolding mixture of this embodiment may then be incubated. In oneembodiment, the refolding mixture is incubated at room temperature forfour to twenty-four hours. The reduced and denatured insulin polypeptidemixture may then be further isolated or purified.

As stated herein, the pH of the first insulin polypeptide mixture may beadjusted prior to performing any subsequent isolation steps. Inaddition, the first insulin polypeptide mixture or any subsequentmixture thereof may be concentrated using techniques known in the art.Moreover, the elution buffer comprising the first insulin polypeptidemixture or any subsequent mixture thereof may be exchanged for a buffersuitable for the next isolation step using techniques known to those ofordinary skill in the art.

Ion Exchange Chromatography In one embodiment, and as an optional,additional step, ion exchange chromatography may be performed on thefirst insulin polypeptide mixture. See generally ION EXCHANGECHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-1114-21, AmershamBiosciences (Piscataway, N.J.)). Commercially available ion exchangecolumns include HITRAP®, HIPREP®, and HILOAD® Columns (AmershamBiosciences, Piscataway, N.J.). Such columns utilize strong anionexchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® HighPerformance, and Q SEPHAROSE® XL; strong cation exchangers such as SPSEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow, and SP SEPHAROSE®XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow; and weakcation exchangers such as CM SEPHAROSE® Fast Flow (Amersham Biosciences,Piscataway, N.J.). Anion or cation exchange column chromatography may beperformed on the insulin polypeptide at any stage of the purificationprocess to isolate substantially purified insulin polypeptide. Thecation exchange chromatography step may be performed using any suitablecation exchange matrix. Useful cation exchange matrices include, but arenot limited to, fibrous, porous, non-porous, microgranular, beaded, orcross-linked cation exchange matrix materials. Such cation exchangematrix materials include, but are not limited to, cellulose, agarose,dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether, orcomposites of any of the foregoing.

The cation exchange matrix may be any suitable cation exchangerincluding strong and weak cation exchangers. Strong cation exchangersmay remain ionized over a wide pH range and thus, may be capable ofbinding insulin over a wide pH range. Weak cation exchangers, however,may lose ionization as a function of pH. For example, a weak cationexchanger may lose charge when the pH drops below about pH 4 or pH 5.Suitable strong cation exchangers include, but are not limited to,charged functional groups such as sulfopropyl (SP), methyl sulfonate(S), or sulfoethyl (SE). The cation exchange matrix may be a strongcation exchanger, preferably having an insulin binding pH range of about2.5 to about 6.0. Alternatively, the strong cation exchanger may have aninsulin binding pH range of about pH 2.5 to about pH 5.5. The cationexchange matrix may be a strong cation exchanger having an insulinbinding pH of about 3.0. Alternatively, the cation exchange matrix maybe a strong cation exchanger, preferably having an insulin binding pHrange of about 6.0 to about 8.0. The cation exchange matrix may be astrong cation exchanger preferably having an insulin binding pH range ofabout 8.0 to about 12.5. Alternatively, the strong cation exchanger mayhave an insulin binding pH range of about pH 8.0 to about pH 12.0.

Prior to loading the insulin, the cation exchange matrix may beequilibrated, for example, using several column volumes of a dilute,weak acid, e.g., four column volumes of 20 mM acetic acid, pH 3.Following equilibration, the insulin may be added and the column may bewashed one to several times, prior to elution of substantially purifiedinsulin, also using a weak acid solution such as a weak acetic acid orphosphoric acid solution. For example, approximately 2-4 column volumesof 20 mM acetic acid, pH 3, may be used to wash the column. Additionalwashes using, e.g., 2-4 column volumes of 0.05 M sodium acetate, pH 5.5,or 0.05 M sodium acetate mixed with 0.1 M sodium chloride, pH 5.5, mayalso be used. Alternatively, using methods known in the art, the cationexchange matrix may be equilibrated using several column volumes of adilute, weak base.

Alternatively, substantially purified insulin may be eluted bycontacting the cation exchanger matrix with a buffer having asufficiently low pH or ionic strength to displace the insulin from thematrix. The pH of the elution buffer may range from about pH 2.5 toabout pH 6.0. More specifically, the pH of the elution buffer may rangefrom about pH 2.5 to about pH 5.5, about pH 2.5 to about pH 5.0. Theelution buffer may have a pH of about 3.0. In addition, the quantity ofelution buffer may vary widely and will generally be in the range ofabout 2 to about 10 column volumes.

Following adsorption of the insulin polypeptide to the cation exchangermatrix, substantially purified insulin polypeptide may be eluted bycontacting the matrix with a buffer having a sufficiently high pH orionic strength to displace the insulin polypeptide from the matrix.Suitable buffers for use in high pH elution of substantially purifiedinsulin polypeptide may include, but not limited to, citrate, phosphate,formate, acetate, HEPES, and MES buffers ranging in concentration fromat least about 5 mM to at least about 100 mM.

Reverse-Phase Chromatography RP-HPLC may be performed to purify proteinsfollowing suitable protocols that are known to those of ordinary skillin the art. See, e.g., Pearson et al., ANAL BIOCHEM. (1982) 124:217-230(1982); Rivier et al., J. CHROM. (1983) 268:112-119; Kunitani et al., J.CHROM. (1986) 359:391-402. RP-HPLC may be performed on the insulinpolypeptide to isolate substantially purified insulin polypeptide. Inthis regard, silica derivatized resins with alkyl functionalities with awide variety of lengths, including, but not limited to, at least aboutC3 to at least about C30, at least about C3 to at least about C20, or atleast about C3 to at least about C18, resins may be used. Alternatively,a polymeric resin may be used. For example, TosoHaas AmberchromeCG1000sd resin may be used, which is a styrene polymer resin. Cyano orpolymeric resins with a wide variety of alkyl chain lengths may also beused. Furthermore, the RP-HPLC column may be washed with a solvent suchas ethanol. The Source RP column is another example of a RP-HPLC column.

A suitable elution buffer containing an ion pairing agent and an organicmodifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile orethanol, may be used to elute the insulin polypeptide from the RP-HPLCcolumn. The most commonly used ion pairing agents include, but are notlimited to, acetic acid, formic acid, perchloric acid, phosphoric acid,trifluoroacetic acid, heptafluorobutyric acid, triethylamine,tetramethylammonium, tetrabutylammonium, and triethylammonium acetate.Elution may be performed using one or more gradients or isocraticconditions, with gradient conditions preferred to reduce the separationtime and to decrease peak width. Another method involves the use of twogradients with different solvent concentration ranges. Examples ofsuitable elution buffers for use herein may include, but are not limitedto, ammonium acetate and acetonitrile solutions.

Hydrophobic Interaction Chromatography Purification TechniquesHydrophobic interaction chromatography (HIC) may be performed on theinsulin polypeptide. See generally HYDROPHOBIC INTERACTIONCHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No, 18-1020-90,Amersham Biosciences (Piscataway, N.J.) which is incorporated byreference herein. Suitable HIC matrices may include, but are not limitedto, alkyl- or aryl-substituted matrices, such as butyl-, hexyl-, octyl-or phenyl-substituted matrices including agarose, cross-linked agarose,sepharose, cellulose, silica, dextran, polystyrene, poly(methacrylate)matrices, and mixed mode resins, including but not limited to, apolyethyleneamine resin or a butyl- or phenyl-substitutedpoly(methacrylate) matrix. Commercially available sources forhydrophobic interaction column chromatography include, but are notlimited to, HITRAP®, HIPREP®, and HILOAD® columns (Amersham Biosciences,Piscataway, N.J.).

Briefly, prior to loading, the HIC column may be equilibrated usingstandard buffers known to those of ordinary skill in the art, such as anacetic acid/sodium chloride solution or HEPES containing ammoniumsulfate. Ammonium sulfate may be used as the buffer for loading the HICcolumn. After loading the insulin polypeptide, the column may thenwashed using standard buffers and conditions to remove unwantedmaterials but retaining the insulin polypeptide on the HIC column. Theinsulin polypeptide may be eluted with about 3 to about 10 columnvolumes of a standard buffer, such as a HEPES buffer containing EDTA andlower ammonium sulfate concentration than the equilibrating buffer, oran acetic acid/sodium chloride buffer, among others. A decreasing linearsalt gradient using, for example, a gradient of potassium phosphate, mayalso be used to elute the insulin molecules. The eluant may then beconcentrated, for example, by filtration such as diafiltration orultrafiltration. Diafiltration may be utilized to remove the salt usedto elute the insulin polypeptide.

Other Purification Techniques Yet another isolation step using, forexample, gel filtration (GEL FILTRATION; PRINCIPLES AND METHODS (Cat.No. 18-1022-18, Amersham Biosciences, Piscataway, N.J.) which isincorporated by reference herein, hydroxyapatite chromatography(suitable matrices include, but are not limited to, HA-Ultrogel, HighResolution (Calbiochem), CHT Ceramic Hydroxyapatite (BioRad), Bio-GelHTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption,ultrafiltration, diafiltration, lyophilization, and the like, may beperformed on the first insulin polypeptide mixture or any subsequentmixture thereof, to remove any excess salts and to replace the bufferwith a suitable buffer for the next isolation step or even formulationof the final drug product.

The yield of insulin polypeptide, including substantially purifiedinsulin polypeptide, may be monitored at each step described hereinusing techniques known to those of ordinary skill in the art. Suchtechniques may also be used to assess the yield of substantiallypurified insulin polypeptide following the last isolation step. Forexample, the yield of insulin polypeptide may be monitored using any ofseveral reverse phase high pressure liquid chromatography columns,having a variety of alkyl chain lengths such as cyano RP-HPLC,C18RP-HPLC; as well as cation exchange HPLC and gel filtration HPLC.

In specific embodiments of the present invention, the yield of insulinafter each purification step may be at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, at least about 99%, at least about99.9%, or at least about 99.99%, of the insulin in the starting materialfor each purification step.

Purity may be determined using standard techniques, such as SDS-PAGE, orby measuring insulin polypeptide using Western blot and ELISA assays.For example, polyclonal antibodies may be generated against proteinsisolated from negative control yeast fermentation and the cationexchange recovery. The antibodies may also be used to probe for thepresence of contaminating host cell proteins.

RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles, thesurfaces of which carry C4-alkyl chains. The separation of insulinpolypeptide from the proteinaceous impurities is based on differences inthe strength of hydrophobic interactions. Elution is performed with anacetonitrile gradient in diluted trifluoroacetic acid. Preparative HPLCis performed using a stainless steel column (filled with 2.8 to 3.2liter of Vydac C4 silicagel). The Hydroxyapatite Ultrogel eluate isacidified by adding trifluoroacetic acid and loaded onto the Vydac C4column. For washing and elution an acetonitrile gradient in dilutedtrifluoroacetic acid is used. Fractions are collected and immediatelyneutralized with phosphate buffer. The insulin polypeptide fractionswhich are within the IPC limits are pooled.

DEAE Sepharose (Pharmacia) material consists of diethylaminoethyl(DEAE)-groups which are covalently bound to the surface of Sepharosebeads. The binding of insulin polypeptide to the DEAE groups is mediatedby ionic interactions. Acetonitrile and trifluoroacetic acid passthrough the column without being retained. After these substances havebeen washed off, trace impurities are removed by washing the column withacetate buffer at a low pH. Then the column is washed with neutralphosphate buffer and insulin polypeptide is eluted with a buffer withincreased ionic strength. The column is packed with DEAE Sepharose fastflow. The column volume is adjusted to assure an insulin polypeptideload in the range of 3-10 mg insulin polypeptide/ml gel. The column iswashed with water and equilibration buffer (sodium/potassium phosphate).The pooled fractions of the HPLC eluate are loaded and the column iswashed with equilibration buffer. Then the column is washed with washingbuffer (sodium acetate buffer) followed by washing with equilibrationbuffer. Subsequently, insulin polypeptide is eluted from the column withelution buffer (sodium chloride, sodium/potassium phosphate) andcollected in a single fraction in accordance with the master elutionprofile. The eluate of the DEAE Sepharose column is adjusted to thespecified conductivity. The resulting drug substance is sterile filteredinto Teflon bottles and stored at −70° C.

Additional methods that may be employed include, but are not limited to,steps to remove endotoxins. Endotoxins are lipopoly-saccharides (LPSs)which are located on the outer membrane of Gram-negative host cells,such as, for example, Escherichia coli. Methods for reducing endotoxinlevels are known to one of ordinary skill in the art and include, butare not limited to, purification techniques using silica supports, glasspowder or hydroxyapatite, reverse-phase, affinity, size-exclusion,anion-exchange chromatography, hydrophobic interaction chromatography, acombination of these methods, and the like. Modifications or additionalmethods may be required to remove contaminants such as co-migratingproteins from the polypeptide of interest. Methods for measuringendotoxin levels are known to one of ordinary skill in the art andinclude, but are not limited to, Limulus Amebocyte Lysate (LAL) assays.The Endosafe™-PTS assay is a calorimetric, single tube system thatutilizes cartridges preloaded with LAL reagent, chromogenic substrate,and control standard endotoxin along with a handheld spectrophotometer.Alternate methods include, but are not limited to, a Kinetic LAL methodthat is turbidmetric and uses a 96 well format.

A wide variety of methods and procedures can be used to assess the yieldand purity of a insulin protein comprising one or more non-naturallyencoded amino acids, including but not limited to, the Bradford assay,SDS-PAGE, silver stained SDS-PAGE, coomassie stained SDS-PAGE, massspectrometry (including but not limited to, MALDI-TOF) and other methodsfor characterizing proteins known to one of ordinary skill in the art.

Additional methods include, but are not limited to: SDS-PAGE coupledwith protein staining methods, immunoblotting, matrix assisted laserdesorption/ionization-mass spectrometry (MALDI-MS), liquidchromatography/mass spectrometry, isoelectric focusing, analytical anionexchange, chromatofocusing, and circular dichroism.

An in vivo method, termed selective pressure incorporation, wasdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been incorporated in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also beenincorporated efficiently, allowing for additional modification ofproteins by chemical means. See, e.g., J. C. van Hest and D. A. Tirrell,FEBS Lett., 428:68 (1998); J. C. van Hest, K. L. Kiick and D. A.Tirrell, J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A.Tirrell, Tetrahedron, 56:9487 (2000); U.S. Pat. No. 6,586,207; U.S.Patent Publication 2002/0042097, which are incorporated by referenceherein.

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.One way to expand the scope of this method is to relax the substratespecificity of aminoacyl-tRNA synthetases, which has been achieved in alimited number of cases. For example, replacement of Ala²⁹⁴ by Gly inEscherichia coli phenylalanyl-tRNA synthetase (PheRS) increases the sizeof substrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soll andS, Nishimura, J. Biol. Chem., 275:40324 (2000).

Another strategy to incorporate unnatural amino acids into proteins invivo is to modify synthetases that have proofreading mechanisms. Thesesynthetases cannot discriminate and therefore activate amino acids thatare structurally similar to the cognate natural amino acids. This erroris corrected at a separate site, which deacylates the mischarged aminoacid from the tRNA to maintain the fidelity of protein translation. Ifthe proofreading activity of the synthetase is disabled, structuralanalogs that are misactivated may escape the editing function and beincorporated. This approach has been demonstrated recently with thevalyl-tRNA synthetase (ValRS). See, V. Doring, H. D. Mootz, L. A.Nangle, T. L. Hendrickson, V. de Crecy-Lagard, P. Schimmel and P.Marliere, Science, 292:501 (2001). ValRS can misaminoacylate tRNAValwith Cys, Thr, or aminobutyrate (Abu); these noncognate amino acids aresubsequently hydrolyzed by the editing domain. After random mutagenesisof the Escherichia coli chromosome, a mutant Escherichia coli strain wasselected that has a mutation in the editing site of ValRS. Thisedit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abusterically resembles Cys group of Cys is replaced with —CH3 in Abu), themutant ValRS also incorporates Abu into proteins when this mutantEscherichia coli strain is grown in the presence of Abu. Massspectrometric analysis shows that about 24% of valines are replaced byAbu at each valine position in the native protein.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of proteins containing novel amino acids. Forexample, see the following publications and references cited within,which are as follows: Crick, F. H. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,192:1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides.XXXVI. The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am. Chem,88(24):5914-5919 (1966); Kaiser, E. T. Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc ChemRes, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptidesegment coupling catalyzed by the semisynthetic enzyme thiosubtilisin,J. Am. Chem. Soc, 109:3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256(5054):221-225 (1992);Chaiken, I. M. Semisynthetic peptides and proteins, CRC Crit RevBiochem, 11(3):255-301 (1981); Offord, R. E. Protein engineering bychemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D. Y.,Burnier, J., Quan, C., Stanley, M., Torn, J., Wells, J. A. A DesignedPeptide Ligase for Total Synthesis of Ribonuclease A with UnnaturalCatalytic Residues, Science, 266(5183):243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,238(4832):1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E.The chemical modification of enzymatic specificity, Annu Rev Biochem,54:565-595 (1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation ofenyzme active sites, Science, 226(4674):505-511 (1984); Neet, K. E.,Nanci A, Koshland, D. E. Properties of thiol-subtilisin, J. Biol. Chem.,243(24):6392-6401 (1968); Polgar, L. et M. L. Bender. A new enzymecontaining a synthetically formed active site. Thiol-subtilisin. J. Am.Chem Soc, 88:3153-3154 (1966); and, Pollack, S. J., Nakayama, G.Schultz, P. G. Introduction of nucleophiles and spectroscopic probesinto antibody combining sites, Science, 242(4881):1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U. C.,Walter, P., Hohnson, A. E. Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signalrecognition particle, Proc. Natl. Acad. Sci, 83(22):8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid,See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am. Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., vol. 202, 301-336(1992); and, Mendel, D., Cornish, V. W. & Schultz, P. G. Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys. BiomolStruct. 24, 435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′-3′ Exonucleases inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 16(3):791-802 (1988). When the acylated suppressor tRNA andthe mutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; Kobayashi et al., (2003) Nature Structural Biology 10(6):425-432;and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,255(5041):197-200 (1992).

A tRNA may be aminoacylated with a desired amino acid by any method ortechnique, including but not limited to, chemical or enzymaticaminoacylation.

Aminoacylation may be accomplished by aminoacyl tRNA synthetases or byother enzymatic molecules, including but not limited to, ribozymes. Theterm “ribozyme” is interchangeable with “catalytic RNA.” Cech andcoworkers (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987,Concepts Biochem. 64:221-226) demonstrated the presence of naturallyoccurring RNAs that can act as catalysts (ribozymes). However, althoughthese natural RNA catalysts have only been shown to act on ribonucleicacid substrates for cleavage and splicing, the recent development ofartificial evolution of ribozymes has expanded the repertoire ofcatalysis to various chemical reactions. Studies have identified RNAmolecules that can catalyze aminoacyl-RNA bonds on their own(2′)3′-termini (Illangakekare et al., 1995 Science 267:643-647), and anRNA molecule which can transfer an amino acid from one RNA molecule toanother (Lohse et al., 1996, Nature 381:442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporatedby reference herein, describes methods to construct ribozymes and theiruse in aminoacylation of tRNAs with naturally encoded and non-naturallyencoded amino acids. Substrate-immobilized forms of enzymatic moleculesthat can aminoacylate tRNAs, including but not limited to, ribozymes,may enable efficient affinity purification of the aminoacylatedproducts. Examples of suitable substrates include agarose, sepharose,and magnetic beads. The production and use of a substrate-immobilizedform of ribozyme for aminoacylation is described in Chemistry andBiology 2003, 10:1077-1084 and U.S. Patent Application Publication2003/0228593, which are incorporated by reference herein.

Chemical aminoacylation methods include, but are not limited to, thoseintroduced by Hecht and coworkers (Hecht, S. M. Ace. Chem. Res. 1992,25, 545; Heckler, T. G.; Roesser, J. R.; Xu, C.; Chang, P.; Hecht, S. M.Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L.; Kuroda, Y.;Kitano, S. J. Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin,Dougherty and others (Cornish, V. W.; Mendel, D.; Schultz, P. G. Anew.Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, S. A.; Ellman, J. A.;Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722; Noren, C. J.;Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989,244, 182; Bain, J. D.; Glabe, C. G.; Dix, T. A.; Chamberlin, A. R. J.Am. Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biol. 1997,4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W.et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996,271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), whichare incorporated by reference herein, to avoid the use of synthetases inaminoacylation. Such methods or other chemical aminoacylation methodsmay be used to aminoacylate tRNA molecules.

Methods for generating catalytic RNA may involve generating separatepools of randomized ribozyme sequences, performing directed evolution onthe pools, screening the pools for desirable aminoacylation activity,and selecting sequences of those ribozymes exhibiting desiredaminoacylation activity.

Ribozymes can comprise motifs and/or regions that facilitate acylationactivity, such as a GGU motif and a U-rich region. For example, it hasbeen reported that U-rich regions can facilitate recognition of an aminoacid substrate, and a GGU-motif can form base pairs with the 3′ terminiof a tRNA. In combination, the GGU and motif and U-rich regionfacilitate simultaneous recognition of both the amino acid and tRNAsimultaneously, and thereby facilitate aminoacylation of the 3′ terminusof the tRNA.

Ribozymes can be generated by in vitro selection using a partiallyrandomized r24mini conjugated with tRNA^(Asn) _(CCCG), followed bysystematic engineering of a consensus sequence found in the activeclones. An exemplary ribozyme obtained by this method is termed “Fx3ribozyme” and is described in U.S. Pub. App. No. 2003/0228593, thecontents of which is incorporated by reference herein, acts as aversatile catalyst for the synthesis of various aminoacyl-tRNAs chargedwith cognate non-natural amino acids.

Immobilization on a substrate may be used to enable efficient affinitypurification of the aminoacylated tRNAs. Examples of suitable substratesinclude, but are not limited to, agarose, sepharose, and magnetic beads.Ribozymes can be immobilized on resins by taking advantage of thechemical structure of RNA, such as the 3′-cis-diol on the ribose of RNAcan be oxidized with periodate to yield the corresponding dialdehyde tofacilitate immobilization of the RNA on the resin. Various types ofresins can be used including inexpensive hydrazide resins whereinreductive amination makes the interaction between the resin and theribozyme an irreversible linkage. Synthesis of aminoacyl-tRNAs can besignificantly facilitated by this on-column aminoacylation technique.Kourouklis et al. Methods 2005; 36:239-4 describe a column-basedaminoacylation system.

Isolation of the aminoacylated tRNAs can be accomplished in a variety ofways. One suitable method is to elute the aminoacylated tRNAs from acolumn with a buffer such as a sodium acetate solution with 10 mM EDTA,a buffer containing 50 mMN-(2-hydroxyethyl)piperazine-N′-(3-propanesulfonic acid), 12.5 mM KCl,pH 7.0, 10 mM EDTA, or simply an EDTA buffered water (pH 7.0).

The aminoacylated tRNAs can be added to translation reactions in orderto incorporate the amino acid with which the tRNA was aminoacylated in aposition of choice in a polypeptide made by the translation reaction.Examples of translation systems in which the aminoacylated tRNAs of thepresent invention may be used include, but are not limited to celllysates. Cell lysates provide reaction components necessary for in vitrotranslation of a polypeptide from an input mRNA. Examples of suchreaction components include but are not limited to ribosomal proteins,rRNA, amino acids, tRNAs, GTP, ATP, translation initiation andelongation factors and additional factors associated with translation.Additionally, translation systems may be batch translations orcompartmentalized translation. Batch translation systems combinereaction components in a single compartment while compartmentalizedtranslation systems separate the translation reaction components fromreaction products that can inhibit the translation efficiency. Suchtranslation systems are available commercially.

Further, a coupled transcription/translation system may be used. Coupledtranscription/translation systems allow for both transcription of aninput DNA into a corresponding mRNA, which is in turn translated by thereaction components. An example of a commercially available coupledtranscription/translation is the Rapid Translation System (RTS, RocheInc.), The system includes a mixture containing E. coli lysate forproviding translational components such as ribosomes and translationfactors. Additionally, an RNA polymerase is included for thetranscription of the input DNA into an mRNA template for use intranslation. RTS can use compartmentalization of the reaction componentsby way of a membrane interposed between reaction compartments, includinga supply/waste compartment and a transcription/translation compartment.

Aminoacylation of tRNA may be performed by other agents, including butnot limited to, transferases, polymerases, catalytic antibodies,multi-functional proteins, and the like.

Stephan in Scientist 2005 Oct. 10; pages 30-33 describes additionalmethods to incorporate non-naturally encoded amino acids into proteins.Lu et al. in Mol. Cell. 2001 October; 8(4):759-69 describe a method inwhich a protein is chemically ligated to a synthetic peptide containingunnatural amino acids (expressed protein ligation).

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Thong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P, M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers a wide variety of advantages including but not limitedto, high yields of mutant proteins, technical ease, the potential tostudy the mutant proteins in cells or possibly in living organisms andthe use of these mutant proteins in therapeutic treatments anddiagnostic uses. The ability to include unnatural amino acids withvarious sizes, acidities, nucleophilicities, hydrophobicities, and otherproperties into proteins can greatly expand our ability to rationallyand systematically manipulate the structures of proteins, both to probeprotein function and create new proteins or organisms with novelproperties.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998).

It may also be possible to obtain expression of a insulin polynucleotideof the present invention using a cell-free (in-vitro) translationalsystem. Translation systems may be cellular or cell-free, and may beprokaryotic or eukaryotic. Cellular translation systems include, but arenot limited to, whole cell preparations such as permeabilized cells orcell cultures wherein a desired nucleic acid sequence can be transcribedto mRNA and the mRNA translated. Cell-free translation systems arecommercially available and many different types and systems arewell-known. Examples of cell-free systems include, but are not limitedto, prokaryotic lysates such as Escherichia coli lysates, and eukaryoticlysates such as wheat germ extracts, insect cell lysates, rabbitreticulocyte lysates, rabbit oocyte lysates and human cell lysates.Eukaryotic extracts or lysates may be preferred when the resultingprotein is glycosylated, phosphorylated or otherwise modified becausemany such modifications are only possible in eukaryotic systems. Some ofthese extracts and lysates are available commercially (Promega; Madison,Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.;GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, such as the caninepancreatic extracts containing microsomal membranes, are also availablewhich are useful for translating secretory proteins. In these systems,which can include either mRNA as a template (in-vitro translation) orDNA as a template (combined in-vitro transcription and translation), thein vitro synthesis is directed by the ribosomes. Considerable effort hasbeen applied to the development of cell-free protein expression systems.See, e.g., Kim, D. M. and J. R. Swartz, Biotechnology andBioengineering, 74 :309-316 (2001); Kim, D. M. and J. R. Swartz,Biotechnology Letters, 22, 1537-1542, (2000); Kim, D. M., and J. R.Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D. M., and J.R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999); andPatnaik, R. and J. R. Swartz, Biotechniques 24, 862-868, (1998); U.S.Pat. No. 6,337,191; U.S. Patent Publication No. 200210081660; WO00/55353; WO 90/05785, which are incorporated by reference herein.Another approach that may be applied to the expression of insulinpolypeptides comprising a non-naturally encoded amino acid includes themRNA-peptide fusion technique. See, e.g., R. Roberts and J. Szostak,Proc. Natl. Acad. Sci. (USA) 94:12297-12302 (1997); A. Frankel, et al.,Chemistry & Biology 10:1043-1050 (2003). In this approach, an mRNAtemplate linked to puromycin is translated into peptide on the ribosome.If one or more tRNA molecules has been modified, non-natural amino acidscan be incorporated into the peptide as well. After the last mRNA codonhas been read, puromycin captures the C-terminus of the peptide. If theresulting mRNA-peptide conjugate is found to have interesting propertiesin an in vitro assay, its identity can be easily revealed from the mRNAsequence. In this way, one may screen libraries of insulin polypeptidescomprising one or more non-naturally encoded amino acids to identifypolypeptides having desired properties. More recently, in vitro ribosometranslations with purified components have been reported that permit thesynthesis of peptides substituted with non-naturally encoded aminoacids. See, e.g., A. Forster et al., Proc. Natl. Acad. Sci. (USA)100:6353 (2003).

Reconstituted translation systems may also be used. Mixtures of purifiedtranslation factors have also been used successfully to translate mRNAinto protein as well as combinations of lysates or lysates supplementedwith purified translation factors such as initiation factor-1 (IF-1),IF-2, IF-3 (α or β), elongation factor T (EF-Tu), or terminationfactors. Cell-free systems may also be coupled transcription/translationsystems wherein DNA is introduced to the system, transcribed into mRNAand the mRNA translated as described in Current Protocols in MolecularBiology (F. M. Ausubel et al. editors, Wiley Interscience, 1993), whichis hereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(hnRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

Macromolecular Polymers Coupled to Insulin Polypeptides

Various modifications to the non-natural amino acid polypeptidesdescribed herein can be effected using the compositions, methods,techniques and strategies described herein. These modifications includethe incorporation of further functionality onto the non-natural aminoacid component of the polypeptide, including but not limited to, alabel; a dye; a polymer; a water-soluble polymer; a derivative ofpolyethylene glycol; a photocrosslinker; radionuclide; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide; a water-soluble dendrimer; acyclodextrin; an inhibitory ribonucleic acid; a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; anactinic radiation excitable moiety; a photoisomerizable moiety; biotin;a derivative of biotin; a biotin analogue; a moiety incorporating aheavy atom; a chemically cleavable group; a photocleavable group; anelongated side chain; a carbon-linked sugar; a redox-active agent; anamino thioacid; a toxic moiety; an isotopically labeled moiety; abiophysical probe; a phosphorescent group; a chemiluminescent group; anelectron dense group; a magnetic group; an intercalating group; achromophore; an energy transfer agent; a biologically active agent; adetectable label; a small molecule; a quantum dot; a nanotransmitter; aradionucleotide; a radiotransmitter; a neutron-capture agent; or anycombination of the above, or any other desirable compound or substance.As an illustrative, non-limiting example of the compositions, methods,techniques and strategies described herein, the following descriptionwill focus on adding macromolecular polymers to the non-natural aminoacid polypeptide with the understanding that the compositions, methods,techniques and strategies described thereto are also applicable (withappropriate modifications, if necessary and for which one of skill inthe art could make with the disclosures herein) to adding otherfunctionalities, including but not limited to those listed above.

A wide variety of macromolecular polymers and other molecules can belinked to insulin polypeptides of the present invention to modulatebiological properties of the insulin polypeptide, and/or provide newbiological properties to the insulin molecule. These macromolecularpolymers can be linked to the Insulin polypeptide via a naturallyencoded amino acid, via a non-naturally encoded amino acid, or anyfunctional substituent of a natural or non-natural amino acid, or anysubstituent or functional group added to a natural or non-natural aminoacid. The molecular weight of the polymer may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. The molecular weight of the polymer may be between about 100 Daand about 100,000 Da, including but not limited to, 100,000 Da, 95,000Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da,60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da,7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da,900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100Da. In some embodiments, the molecular weight of the polymer is betweenabout 100 Da and about 50,000 Da. In some embodiments, the molecularweight of the polymer is between about 100 Da and about 40,000 Da. Insome embodiments, the molecular weight of the polymer is between about1,000 Da and about 40,000 Da. In some embodiments, the molecular weightof the polymer is between about 5,000 Da and about 40,000 Da. In someembodiments, the molecular weight of the polymer is between about 10,000Da and about 40,000 Da.

The present invention provides substantially homogenous preparations ofpolymer:protein conjugates. “Substantially homogenous” as used hereinmeans that polymer:protein conjugate molecules are observed to begreater than half of the total protein. The polymer:protein conjugatehas biological activity and the present “substantially homogenous”PEGylated insulin polypeptide preparations provided herein are thosewhich are homogenous enough to display the advantages of a homogenouspreparation, e.g., ease in clinical application in predictability of lotto lot pharmacokinetics.

One may also choose to prepare a mixture of polymer:protein conjugatemolecules, and the advantage provided herein is that one may select theproportion of mono-polymer:protein conjugate to include in the mixture.Thus, if desired, one may prepare a mixture of various proteins withvarious numbers of polymer moieties attached (i.e., di-, tri-, tetra-,etc.) and combine said conjugates with the mono-polymer:proteinconjugate prepared using the methods of the present invention, and havea mixture with a predetermined proportion of mono-polymer:proteinconjugates.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.For therapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable.

Examples of polymers include but are not limited to polyalkyl ethers andalkoxy-capped analogs thereof (e.g., polyoxyethylene glycol,polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogsthereof, especially polyoxyethylene glycol, the latter is also known aspolyethyleneglycol or PEG); polyvinylpyrrolidones; polyvinylalkylethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyloxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkylacrylamides (e.g., polyhydroxypropylmethacrylamide and derivativesthereof); polyhydroxyalkyl acrylates; polysialic acids and analogsthereof; hydrophilic peptide sequences; polysaccharides and theirderivatives, including dextran and dextran derivatives, e.g.,carboxymethyldextran, dextran sulfates, aminodextran; cellulose and itsderivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses;chitin and its derivatives, e.g., chitosan, succinyl chitosan,carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and itsderivatives; starches; alginates; chondroitin sulfate; albumin; pullulanand carboxymethyl pullulan; polyaminoacids and derivatives thereof,e.g., polyglutamic acids, polylysines, polyaspartic acids,polyaspartamides; maleic anhydride copolymers such as: styrene maleicanhydride copolymer, divinylethyl ether maleic anhydride copolymer;polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixturesthereof, and derivatives of the foregoing.

The proportion of polyethylene glycol molecules to protein moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

As used herein, and when contemplating PEG: insulin polypeptideconjugates, the term “therapeutically effective amount” refers to anamount which gives the desired benefit to a patient. The amount willvary from one individual to another and will depend upon a number offactors, including the overall physical condition of the patient and theunderlying cause of the condition to be treated. The amount of insulinpolypeptide used for therapy gives an acceptable rate of change andmaintains desired response at a beneficial level. A therapeuticallyeffective amount of the present compositions may be readily ascertainedby one of ordinary skill in the art using publicly available materialsand procedures.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water solublepolymer is a poly(alkylene glycol), such as polyethylene glycol) (PEG),but other water soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods known to those of ordinary skill in the art(Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3,pages 138-161). The term “PEG” is used broadly to encompass anypolyethylene glycol molecule, without regard to size or to modificationat an end of the PEG, and can be represented as linked to the insulinpolypeptide by the formula:

XO—(CH₂CH₂O)_(n)—CH₂CH₂—Y

where n is 2 to 10,000 and X is H or a terminal modification, includingbut not limited to, a C₁₋₄ alkyl, a protecting group, or a terminalfunctional group.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the 20 common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to a insulin polypeptide via a naturally-occurring ornon-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the Insulin polypeptide to form a Huisgen [3+2]cycloaddition product. Alternatively, an alkyne group on the PEG can bereacted with an azide group present in a non-naturally encoded aminoacid to form a similar product. In some embodiments, a strongnucleophile (including but not limited to, hydrazine, hydrazide,hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketonegroup present in a non-naturally encoded amino acid to form a hydrazone,oxime or semicarbazone, as applicable, which in some cases can befurther reduced by treatment with an appropriate reducing agent.Alternatively, the strong nucleophile can be incorporated into theInsulin polypeptide via a non-naturally encoded amino acid and used toreact preferentially with a ketone or aldehyde group present in thewater soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). The molecular weight of PEG may be of a wide range,including but not limited to, between about 100 Da and about 100,000 Daor more. PEG may be between about 100 Da and about 100,000 Da, includingbut not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da,45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, PEG isbetween about 100 Da and about 50,000 Da. In some embodiments, PEG isbetween about 100 Da and about 40,000 Da. In some embodiments, PEG isbetween about 1,000 Da and about 40,000 Da. In some embodiments, PEG isbetween about 5,000 Da and about 40,000 Da. In some embodiments, PEG isbetween about 10,000 Da and about 40,000 Da. Branched chain PEGs,including but not limited to, PEG molecules with each chain having a MWranging from 1-100 kDa (including but not limited to, 1-50 kDa or 5-20kDa) can also be used. The molecular weight of each chain of thebranched chain PEG may be, including but not limited to, between about1,000 Da and about 100,000 Da or more. The molecular weight of eachchain of the branched chain PEG may be between about 1,000 Da and about100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da,55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In someembodiments, the molecular weight of each chain of the branched chainPEG is between about 1,000 Da and about 50,000 Da. In some embodiments,the molecular weight of each chain of the branched chain PEG is betweenabout 1,000 Da and about 40,000 Da. In some embodiments, the molecularweight of each chain of the branched chain PEG is between about 5,000 Daand about 40,000 Da. In some embodiments, the molecular weight of eachchain of the branched chain PEG is between about 5,000 Da and about20,000 Da. A wide range of PEG molecules are described in, including butnot limited to, the Shearwater Polymers, Inc. catalog, NektarTherapeutics catalog, incorporated herein by reference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2] cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the Insulin polypeptide variant with a PEGderivative contains a chemical functionality that is reactive with thechemical functionality present on the side chain of the non-naturallyencoded amino acid.

The invention provides in some embodiments azide- andacetylene-containing polymer derivatives comprising a water solublepolymer backbone having an average molecular weight from about 800 Da toabout 100,000 Da. The polymer backbone of the water-soluble polymer canbe poly(ethylene glycol). However, it should be understood that a widevariety of water soluble polymers including but not limited topoly(ethylene)glycol and other related polymers, including poly(dextran)and polypropylene glycol), are also suitable for use in the practice ofthis invention and that the use of the term PEG or poly(ethylene glycol)is intended to encompass and include all such molecules. The term PEGincludes, but is not limited to, poly(ethylene glycol) in any of itsforms, including bifunctional PEG, multiarmed PEG, derivatized PEG,forked PEG, branched PEG, pendent PEG (i.e. PEG or related polymershaving one or more functional groups pendent to the polymer backbone),or PEG with degradable linkages therein.

PEG is typically clear, colorless, odorless, soluble in water, stable toheat, inert to many chemical agents, does not hydrolyze or deteriorate,and is generally non-toxic. Poly(ethylene glycol) is considered to bebiocompatible, which is to say that PEG is capable of coexistence withliving tissues or organisms without causing harm. More specifically, PEGis substantially non-immunogenic, which is to say that PEG does not tendto produce an immune response in the body. When attached to a moleculehaving some desirable function in the body, such as a biologicallyactive agent, the PEG tends to mask the agent and can reduce oreliminate any immune response so that an organism can tolerate thepresence of the agent. PEG conjugates tend not to produce a substantialimmune response or cause clotting or other undesirable effects. PEGhaving the formula —CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—, where n is from about3 to about 4000, typically from about 20 to about 2000, is suitable foruse in the present invention. PEG having a molecular weight of fromabout 800 Da to about 100,000 Da are in some embodiments of the presentinvention particularly useful as the polymer backbone. The molecularweight of PEG may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more. The molecular weightof PEG may be between about 100 Da and about 100,000 Da, including butnot limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da,75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da,10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da,3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da,400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecularweight of PEG is between about 100 Da and about 50,000 Da. In someembodiments, the molecular weight of PEG is between about 100 Da andabout 40,000 Da. In some embodiments, the molecular weight of PEG isbetween about 1,000 Da and about 40,000 Da. In some embodiments, themolecular weight of PEG is between about 5,000 Da and about 40,000 Da.In some embodiments, the molecular weight of PEG is between about 10,000Da and about 40,000 Da.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:

—PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG-

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the forms knownin the art including but not limited to those disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da. Themolecular weight of each chain of the polymer backbone may be betweenabout 100 Da and about 100,000 Da, including but not limited to, 100,000Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da,65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da,8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da,1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200Da, and 100 Da. In some embodiments, the molecular weight of each chainof the polymer backbone is between about 100 Da and about 50,000 Da. Insome embodiments, the molecular weight of each chain of the polymerbackbone is between about 100 Da and about 40,000 Da. In someembodiments, the molecular weight of each chain of the polymer backboneis between about 1,000 Da and about 40,000 Da. In some embodiments, themolecular weight of each chain of the polymer backbone is between about5,000 Da and about 40,000 Da. In some embodiments, the molecular weightof each chain of the polymer backbone is between about 10,000 Da andabout 40,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:

X-A-POLY-B-N═N═N

wherein:N═N═N is an azide moiety;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen orsulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462; 5,643,575; andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is merely illustrative, and that all linking moieties having thequalities described above are contemplated to be suitable for use in thepresent invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those of ordinary skill in the art, theselected X moiety should be compatible with the azide group so thatreaction with the azide group does not occur. The azide-containingpolymer derivatives may be homobifunctional, meaning that the secondfunctional group (i.e., X) is also an azide moiety, orheterobifunctional, meaning that the second functional group is adifferent functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zalipsky et at Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zalipsky Eds., ACS, Washington, D.C.,1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (See,e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Makromol, Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. J.Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Biotechnology (NY)8:343 (1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═N

wherein:X is a functional group as described above; andn is about 20 to about 4000.In another embodiment, the polymer derivatives of the invention comprisea polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—W—N═N═N

wherein:W is an aliphatic or aromatic linker moiety comprising between 1-10carbon atoms;n is about 20 to about 4000; andX is a functional group as described above. m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer.

X-PEG-L+N₃ ⁻→X-PEG-N₃

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is polyethylene glycol) and X is afunctional group which does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:

X-PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═N

wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andM is a functional group that is not reactive with the azidefunctionality but that will react efficiently and selectively with the Nfunctional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:

BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure:

X-A-POLY-B-C≡C—R

wherein:R can be either H or an alkyl, alkene, alkyoxy, or aryl or substitutedaryl group;B is a linking moiety, which may be present or absent;POLY is a water-soluble non-antigenic polymer;A is a linking moiety, which may be present or absent and which may bethe same as B or different; andX is a second functional group.

Examples of a linking moiety for A and B include, but are not limited toa multiply-functionalized alkyl group containing up to 18, and maycontain between 1-10 carbon atoms. A heteroatom such as nitrogen, oxygenor sulfur may be included with the alkyl chain. The alkyl chain may alsobe branched at a heteroatom. Other examples of a linking moiety for Aand B include, but are not limited to, a multiply functionalized arylgroup, containing up to 10 and may contain 5-6 carbon atoms. The arylgroup may be substituted with one more carbon atoms, nitrogen, oxygen,or sulfur atoms. Other examples of suitable linking groups include thoselinking groups described in U.S. Pat. Nos. 5,932,462 and 5,643,575 andU.S. Pat. Appl. Publication 2003/0143596, each of which is incorporatedby reference herein. Those of ordinary skill in the art will recognizethat the foregoing list for linking moieties is by no means exhaustiveand is intended to be merely illustrative, and that a wide variety oflinking moieties having the qualities described above are contemplatedto be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:

X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C≡CH

wherein:X is a functional group as described above;n is about 20 to about 4000; andm is between 1 and 10.Specific examples of each of the heterobifunctional PEG polymers areshown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those of ordinary skill in the artand/or disclosed herein. In one method, a water soluble polymer backbonehaving an average molecular weight from about 800 Da to about 100,000Da, the polymer backbone having a first terminus bonded to a firstfunctional group and a second terminus bonded to a suitable nucleophilicgroup, is reacted with a compound that bears both an acetylenefunctionality and a leaving group that is suitable for reaction with thenucleophilic group on the PEG. When the PEG polymer bearing thenucleophilic moiety and the molecule bearing the leaving group arecombined, the leaving group undergoes a nucleophilic displacement and isreplaced by the nucleophilic moiety, affording the desiredacetylene-containing polymer.

X-PEG-Nu+L-A-C→X-PEG-Nu-A-C≡CR′

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via annucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

An exemplary reaction scheme is shown below:

X-PEG-L+—C≡CR′→X-PEG-C≡CR′

wherein:PEG is poly(ethylene glycol) and X is a capping group such as alkoxy ora functional group as described above; andR′ is either H, an alkyl, alkoxy, aryl or aryloxy group or a substitutedalkyl, alkoxyl, aryl or aryloxy group.

In the example above, the leaving group L should be sufficientlyreactive to undergo SN2-type displacement when contacted with asufficient concentration of the acetylene anion. The reaction conditionsrequired to accomplish SN2 displacement of leaving groups by acetyleneanions are known to those of ordinary skill in the art.

Purification of the crude product can usually be accomplished by methodsknown in the art including, but are not limited to, precipitation of theproduct followed by chromatography, if necessary.

Water soluble polymers can be linked to the insulin polypeptides of theinvention. The water soluble polymers may be linked via a non-naturallyencoded amino acid incorporated in the insulin polypeptide or anyfunctional group or substituent of a non-naturally encoded or naturallyencoded amino acid, or any functional group or substituent added to anon-naturally encoded or naturally encoded amino acid. Alternatively,the water soluble polymers are linked to an insulin polypeptideincorporating a non-naturally encoded amino acid via anaturally-occurring amino acid (including but not limited to, cysteine,lysine or the amine group of the N-terminal residue). In some cases, theinsulin polypeptides of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10 non-natural amino acids, wherein one or more non-naturally-encodedamino acid(s) are linked to water soluble polymer(s) (including but notlimited to, PEG and/or oligosaccharides). In some cases, the insulinpolypeptides of the invention further comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more naturally-encoded amino acid(s) linked to water solublepolymers. In some cases, the insulin polypeptides of the inventioncomprise one or more non-naturally encoded amino acid(s) linked to watersoluble polymers and one or more naturally-occurring amino acids linkedto water soluble polymers. In some embodiments, the water solublepolymers used in the present invention enhance the serum half-life ofthe insulin polypeptide relative to the unconjugated form.

The number of water soluble polymers linked to a insulin polypeptide(i.e., the extent of PEGylation or glycosylation) of the presentinvention can be adjusted to provide an altered (including but notlimited to, increased or decreased) pharmacologic, pharmacokinetic orpharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of insulin is increased at least about 10,20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 6-fold, 7-fold,8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold,16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 35-fold,40-fold, 50-fold, or at least about 100-fold over an unmodifiedpolypeptide.

Peg Derivatives Containing a Strong Nucleophilic Group (i.e., Hydrazide,Hydrazine, Hydroxylamine or Semicarbazide)

In one embodiment of the present invention, an insulin polypeptidecomprising a carbonyl-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal hydrazine,hydroxylamine, hydrazide or semicarbazide moiety that is linked directlyto the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure:

RO—(CH2CH2O)n-O—(CH2)m-O—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:

RO—(CH2CH2O)n-O—(CH2)m-X—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure:

RO—(CH2CH2)n-O—(CH2)m-NH—C(O)—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, an insulin polypeptidecomprising a carbonyl-containing amino acid is modified with a PEGderivative that contains a terminal hydroxylamine, hydrazide, hydrazine,or semicarbazide moiety that is linked to the PEG backbone by means ofan amide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure:

RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)(CH2)m-O—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:

RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)(CH2)m-X—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is100-1,000 and X is optionally a carbonyl group (C═O) that can be presentor absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure:

RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)(CH2)m-NH—C(O)—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, an insulin polypeptidecomprising a carbonyl-containing amino acid is modified with a branchedPEG derivative that contains a terminal hydrazine, hydroxylamine,hydrazide or semicarbazide moiety, with each chain of the branched PEGhaving a MW ranging from 10-40 kDa and, may be from 5-20 kDa.

In another embodiment of the invention, an insulin polypeptidecomprising a non-naturally encoded amino acid is modified with a PEGderivative having a branched structure. For instance, in someembodiments, the hydrazine- or hydrazide-terminal PEG derivative willhave the following structure:

[RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)]2CH(CH2)m-X—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:

[RO—(CH2CH2O)n-O—(CH2)2-C(O)—NH—CH2-CH2]2CH—X—(CH2)m-NH—C(O)—NH—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:

[RO—(CH2CH2O)n-O—(CH2)2-C(O)—NH—CH2-CH2]2CH—X—(CH2)m-O—NH2

where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe insulin polypeptide can modulate the binding of the insulinpolypeptide to the insulin polypeptide receptor. In some embodiments,the linkages are arranged such that the insulin polypeptide binds theinsulin polypeptide receptor with a Kd of about 400 nM or lower, with aKd of 150 nM or lower, and in some cases with a Kd of 100 nM or lower,as measured by an equilibrium binding assay, such as that described inSpencer et al., J. Biol. Chem., 263:7862-7867 (1988).

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macromol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) of insulinpolypeptides containing a non-naturally encoded amino acid, such asp-azido-L-phenylalanine, is carried out by any convenient method. Forexample, insulin polypeptide is PEGylated with an alkyne-terminated mPEGderivative. Briefly, an excess of solid mPEG(5000)-O—CH2-C

CH is added, with stirring, to an aqueous solution ofp-azido-L-Phe-containing insulin polypeptide at room temperature.Typically, the aqueous solution is buffered with a buffer having a pKanear the pH at which the reaction is to be carried out (generally aboutpH 4-10). Examples of suitable buffers for PEGylation at pH 7.5, forinstance, include, but are not limited to, HEPES, phosphate, borate,TRIS-HCl, EPPS, and TES. The pH is continuously monitored and adjustedif necessary. The reaction is typically allowed to continue for betweenabout 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated insulin polypeptidevariants from free mPEG(5000)-O—CH2-C≡CH and any high-molecular weightcomplexes of the pegylated insulin polypeptide which may form whenunblocked PEG is activated at both ends of the molecule, therebycrosslinking insulin polypeptide variant molecules. The conditionsduring hydrophobic interaction chromatography are such that freemPEG(5000)-O—CH2-C≡CH flows through the column, while any crosslinkedPEGylated insulin polypeptide variant complexes elute after the desiredforms, which contain one insulin polypeptide variant molecule conjugatedto one or more PEG groups. Suitable conditions vary depending on therelative sizes of the cross-linked complexes versus the desiredconjugates and are readily determined by those of ordinary skill in theart. The eluent containing the desired conjugates is concentrated byultrafiltration and desalted by diafiltration.

If necessary, the PEGylated insulin polypeptide obtained from thehydrophobic chromatography can be purified further by one or moreprocedures known to those of ordinary skill in the art including, butare not limited to, affinity chromatography; anion- or cation-exchangechromatography (using, including but not limited to, DEAE, SEPHAROSE);chromatography on silica; reverse phase HPLC; gel filtration (using,including but not limited to, SEPHADEX G-75); hydrophobic interactionchromatography; size-exclusion chromatography, metal-chelatechromatography; ultrafiltration/diafiltration; ethanol precipitation;ammonium sulfate precipitation; chromatofocusing; displacementchromatography; electrophoretic procedures (including but not limited topreparative isoelectric focusing), differential solubility (includingbut not limited to ammonium sulfate precipitation), or extraction.Apparent molecular weight may be estimated by GPC by comparison toglobular protein standards (Preneta, A Z in PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris & Angal, Eds.) IRL Press 1989,293-306). The purity of the insulin-PEG conjugate can be assessed byproteolytic degradation (including but not limited to, trypsin cleavage)followed by mass spectrometry analysis. Pepinsky R B., et al., J.Pharmcol. & Exp. Ther. 297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of an insulinpolypeptide of the invention can be further derivatized or substitutedwithout limitation.

Azide-Containing PEG Derivatives

In another embodiment of the invention, an insulin polypeptide ismodified with a PEG derivative that contains an azide moiety that willreact with an alkyne moiety present on the side chain of thenon-naturally encoded amino acid. In general, the PEG derivatives willhave an average molecular weight ranging from 1-100 kDa and, in someembodiments, from 10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure:

RO—(CH2CH2O)n-O—(CH2)m-N3

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure:

RO—(CH2CH2O)n-O—(CH2)m-NH—C(O)—(CH2)p-N3

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000 (i.e., average molecular weight is between 5-40kDa).

In another embodiment of the invention, an insulin polypeptidecomprising a alkyne-containing amino acid is modified with a branchedPEG derivative that contains a terminal azide moiety, with each chain ofthe branched PEG having a MW ranging from 10-40 kDa and may be from 5-20kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:

[RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)]2CH(CH2)m-X—(CH2)pN3

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C═O), in each case that can be present or absent.

Alkyne-Containing PEG Derivatives

In another embodiment of the invention, an insulin polypeptide ismodified with a PEG derivative that contains an alkyne moiety that willreact with an azide moiety present on the side chain of thenon-naturally encoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH2CH2O)n-O—(CH2)m-C

CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, an insulin polypeptidecomprising an alkyne-containing non-naturally encoded amino acid ismodified with a PEG derivative that contains a terminal azide orterminal alkyne moiety that is linked to the PEG backbone by means of anamide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure:

RO—(CH2CH2O)n-O—(CH2)m-NH—C(O)—(CH2)p-C

CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, an insulin polypeptidecomprising an azide-containing amino acid is modified with a branchedPEG derivative that contains a terminal alkyne moiety, with each chainof the branched PEG having a MW ranging from 10-40 kDa and may be from5-20 kDa. For instance, in some embodiments, the alkyne-terminal PEGderivative will have the following structure:

[RO—(CH2CH2O)n-O—(CH2)2-NH—C(O)]2CH(CH2)m-X—(CH2)pC

CH

where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10, and n is 100-1,000, and X is optionally an O, N, S or carbonylgroup (C=0), or not present.

Phosphine-Containing PEG Derivatives

In another embodiment of the invention, an insulin polypeptide ismodified with a PEG derivative that contains an activated functionalgroup (including but not limited to, ester, carbonate) furthercomprising an aryl phosphine group that will react with an azide moietypresent on the side chain of the non-naturally encoded amino acid. Ingeneral, the PEG derivatives will have an average molecular weightranging from 1-100 kDa and, in some embodiments, from 10-40 kDa.

In some embodiments, the PEG derivative will have the structure:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

In some embodiments, the PEG derivative will have the structure:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Other PEG Derivatives and General PEGylation Techniques

Other exemplary PEG molecules that may be linked to insulinpolypeptides, as well as PEGylation methods include, but are not limitedto, those described in, e.g., U.S. Patent Publication No. 2004/0001838;2002/0052009; 2003/0162949; 2004/0013637; 2003/0228274; 2003/0220447;2003/0158333; 2003/0143596; 2003/0114647; 2003/0105275; 2003/0105224;2003/0023023; 2002/0156047; 2002/0099133; 2002/0086939; 2002/0082345;2002/0072573; 2002/0052430; 2002/0040076; 2002/0037949; 2002/0002250;2001/0056171; 2001/0044526; 2001/0021763; U.S. Pat. Nos. 6,646,110;5,824,778; 5,476,653; 5,219,564; 5,629,384; 5,736,625; 4,902,502;5,281,698; 5,122,614; 5,473,034; 5,516,673; 5,382,657; 6,552,167;6,610,281; 6,515,100; 6,461,603; 6,436,386; 6,214,966; 5,990,237;5,900,461; 5,739,208; 5,672,662; 5,446,090; 5,808,096; 5,612,460;5,324,844; 5,252,714; 6,420,339; 6,201,072; 6,451,346; 6,306,821;5,559,213; 5,747,646; 5,834,594; 5,849,860; 5,980,948; 6,004,573;6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439 508, WO97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP 809 996,WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 183 503and EP 154 316, which are incorporated by reference herein. Any of thePEG molecules described herein may be used in any form, including butnot limited to, single chain, branched chain, multiarm chain, singlefunctional, bifunctional, multi-functional, or any combination thereof

Additional polymer and PEG derivatives including but not limited to,hydroxylamine (aminooxy) PEG derivatives, are described in the followingpatent applications which are all incorporated by reference in theirentirety herein: U.S. Patent Publication No. 2006/0194256, U.S. PatentPublication No. 2006/0217532, U.S. Patent Publication No. 2006/0217289,U.S. Provisional Patent No. 60/755,338; U.S. Provisional Patent No.60/755,711; U.S. Provisional Patent No. 60/755,018; International PatentApplication No. PCT/US06/49397; WO 2006/069246; U.S. Provisional PatentNo. 60/743,041; U.S. Provisional Patent No. 60/743,040; InternationalPatent Application No. PCT/US06/47822; U.S. Provisional Patent No.60/882,819; U.S. Provisional Patent No. 60/882,500; and U.S. ProvisionalPatent No. 60/870,594.

Heterologous Fc Fusion Proteins

The insulin compounds described above may be fused directly or via apeptide linker to the Fc portion of an immunoglobulin. Immunoglobulinsare molecules containing polypeptide chains held together by disulfidebonds, typically having two light chains and two heavy chains. In eachchain, one domain (V) has a variable amino acid sequence depending onthe antibody specificity of the molecule. The other domains (C) have arather constant sequence common to molecules of the same class.

As used herein, the Fc portion of an immunoglobulin has the meaningcommonly given to the term in the field of immunology. Specifically,this term refers to an antibody fragment which is obtained by removingthe two antigen binding regions (the Fab fragments) from the antibody.One way to remove the Fab fragments is to digest the immunoglobulin withpapain protease. Thus, the Fc portion is formed from approximately equalsized fragments of the constant region from both heavy chains, whichassociate through non-covalent interactions and disulfide bonds. The Fcportion can include the hinge regions and extend through the CH2 and CH3domains to the C-terminus of the antibody. Representative hinge regionsfor human and mouse immunoglobulins can be found in AntibodyEngineering, A Practical Guide, Borrebaeck, C. A. K., ed., W. H. Freemanand Co., 1992, the teachings of which are herein incorporated byreference. The Fc portion can further include one or more glycosylationsites. The amino acid sequences of numerous representative Fc proteinscontaining a hinge region, CH2 and CH3 domains, and one N-glycosylationsite are well known in the art.

There are five types of human immunoglobulin Fc regions with differenteffector functions and pharmacokinetic properties: IgG, IgA, IgM, IgD,and IgE. IgG is the most abundant immunoglobulin in serum. IgG also hasthe longest half-life in serum of any immunoglobulin (23 days). Unlikeother immunoglobulins, IgG is efficiently recirculated following bindingto an Fc receptor. There are four IgG subclasses G1, G2, G3, and G4,each of which has different effector functions. G1, G2, and G3 can bindC1q and fix complement while G4 cannot. Even though G3 is able to bindC1q more efficiently than G1, G1 is more effective at mediatingcomplement-directed cell lysis. G2 fixes complement very inefficiently.The C1q binding site in IgG is located at the carboxy terminal region ofthe CH2 domain.

All IgG subclasses are capable of binding to Fc receptors (CD16, CD32,CD64) with G1 and G3 being more effective than G2 and G4. The Fcreceptor binding region of IgG is formed by residues located in both thehinge and the carboxy terminal regions of the CH2 domain.

IgA can exist both in a monomeric and dimeric form held together by aJ-chain. IgA is the second most abundant Ig in serum, but it has ahalf-life of only 6 days. IgA has three effector functions. It binds toan IgA specific receptor on macrophages and eosinophils, which drivesphagocytosis and degranulation, respectively. It can also fix complementvia an unknown alternative pathway.

IgM is expressed as either a pentamer or a hexamer, both of which areheld together by a J-chain. IgM has a serum half-life of 5 days. Itbinds weakly to C1q via a binding site located in its CH3 domain. IgDhas a half-life of 3 days in serum. It is unclear what effectorfunctions are attributable to this Ig. IgE is a monomeric Ig and has aserum half-life of 2.5 days. IgE binds to two Fc receptors which drivesdegranulation and results in the release of proinflammatory agents.

Depending on the desired in vivo effect, the heterologous fusionproteins of the present invention may contain any of the isotypesdescribed above or may contain mutated Fc regions wherein the complementand/or Fc receptor binding functions have been altered. Thus, theheterologous fusion proteins of the present invention may contain theentire Fc portion of an immunoglobulin, fragments of the Fc portion ofan immunoglobulin, or analogs thereof fused to an interferon betacompound.

The fusion proteins of the present invention can consist of single chainproteins or as multi-chain polypeptides. Two or more Fc fusion proteinscan be produced such that they interact through disulfide bonds thatnaturally form between Fc regions. These multimers can be homogeneouswith respect to the interferon beta compound or they may containdifferent interferon beta compounds fused at the N-terminus of the Fcportion of the fusion protein.

Regardless of the final structure of the fusion protein, the Fc orFc-like region may serve to prolong the in vivo plasma half-life of theinterferon beta compound fused at the N-terminus. Also, the interferonbeta component of a fusion protein compound should retain at least onebiological activity of interferon beta. An increase in therapeutic orcirculating half-life can be demonstrated using the method describedherein or known in the art, wherein the half-life of the fusion proteinis compared to the half-life of the interferon beta compound alone.Biological activity can be determined by in vitro and in vivo methodsknown in the art.

Since the Fc region of IgG produced by proteolysis has the same in vivohalf-life as the intact IgG molecule and Fab fragments are rapidlydegraded, it is believed that the relevant sequence for prolonginghalf-life reside in the CH2 and/or CH3 domains. Further, it has beenshown in the literature that the catabolic rates of IgG variants that donot bind the high-affinity Fc receptor or C1q are indistinguishable fromthe rate of clearance of the parent wild-type antibody, indicating thatthe catabolic site is distinct from the sites involved in Fc receptor orC1q binding. [Wawrzynczak et al., (1992) Molecular Immunology 29:221].Site-directed mutagenesis studies using a murine IgG1 Fc regionsuggested that the site of the IgG1 Fc region that controls thecatabolic rate is located at the CH2-CH3 domain interface. Fc regionscan be modified at the catabolic site to optimize the half-life of thefusion proteins. The Fc region used for the fusion proteins of thepresent invention may be derived from an IgG1 or an IgG4 Fc region, andmay contain both the CH2 and CH3 regions including the hinge region.

Heterologous Albumin Fusion Proteins

Insulin described herein may be fused directly or via a peptide linker,water soluble polymer, or prodrug linker to albumin or an analog,fragment, or derivative thereof. Generally, the albumin proteins thatare part of the fusion proteins of the present invention may be derivedfrom albumin cloned from any species, including human. Human serumalbumin (HSA) consists of a single non-glycosylated polypeptide chain of585 amino acids with a formula molecular weight of 66,500. The aminoacid sequence of human HSA is known [See Meloun, et al. (1975) FEBSLetters 58:136; Behrens, et al. (1975) Fed. Proc. 34:591; Lawn, et al.(1981) Nucleic Acids Research 9:6102-6114; Minghetti, et al. (1986) J.Biol. Chem. 261:6747, each of which are incorporated by referenceherein]. A variety of polymorphic variants as well as analogs andfragments of albumin have been described. [See Weitkamp, et al., (1973)Ann. Hum. Genet. 37:219]. For example, in EP 322,094, various shorterforms of USA. Some of these fragments of HSA are disclosed, includingHSA(1-373), HSA(1-388), HSA(1-389), HSA(1-369), and HSA(1-419) andfragments between 1-369 and 1-419. EP 399,666 discloses albuminfragments that include HSA(1-177) and HSA(1-200) and fragments betweenHSA(1-177) and HSA(1-200).

It is understood that the heterologous fusion proteins of the presentinvention include insulin compounds that are coupled to any albuminprotein including fragments, analogs, and derivatives wherein suchfusion protein is biologically active and has a longer plasma half-lifethan the insulin compound alone. Thus, the albumin portion of the fusionprotein need not necessarily have a plasma half-life equal to that ofnative human albumin. Fragments, analogs, and derivatives are known orcan be generated that have longer half-lives or have half-livesintermediate to that of native human albumin and the insulin compound ofinterest.

The heterologous fusion proteins of the present invention encompassproteins having conservative amino acid substitutions in the insulincompound and/or the Fc or albumin portion of the fusion protein. A“conservative substitution” is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number carbon and heteroatoms in their side chainsdiffers by no more than about four. They have approximately the sameshape when the number of branches in their side chains differs by nomore than one. Amino acids with phenyl or substituted phenyl groups intheir side chains are considered to have about the same size and shape.Except as otherwise specifically provided herein, conservativesubstitutions are preferably made with naturally occurring amino acids.

Wild-type albumin and immunoglobulin proteins can be obtained from avariety of sources. For example, these proteins can be obtained from acDNA library prepared from tissue or cells which express the mRNA ofinterest at a detectable level. Libraries can be screened with probesdesigned using the published DNA or protein sequence for the particularprotein of interest. For example, immunoglobulin light or heavy chainconstant regions are described in Adams, et al. (1980) Biochemistry19:2711-2719; Goughet, et al. (1980) Biochemistry 19:2702-2710; Dolby,et al. (1980) Proc. Natl. Acad. Sci. USA 77:6027-6031; Rice et al.(1982) Proc. Natl. Acad. Sci. USA 79:7862-7862; Falkner, et al. (1982)Nature 298:286-288; and Morrison, et al. (1984) Ann. Rev. Immunol.2:239-256. Some references disclosing albumin protein and DNA sequencesinclude Meloun, et al. (1975) FEBS Letters 58:136; Behrens, et al.(1975) Fed. Proc. 34:591; Lawn, et al. (1981) Nucleic Acids Research9:6102-6114; and Minghetti, et al. (1986) J. Biol. Chem. 261:6747.

Characterization of the Heterologous Fusion Proteins of the PresentInvention

Numerous methods exist to characterize the fusion proteins of thepresent invention. Some of these methods include, but are not limitedto: SDS-PAGE coupled with protein staining methods or immunoblottingusing anti-IgG or anti-HSA antibodies. Other methods include matrixassisted laser desorption/ionization-mass spectrometry (MALDI-MS),liquid chromatography/mass spectrometry, isoelectric focusing,analytical anion exchange, chromatofocusing, and circular dichroism, forexample.

Enhancing Affinity for Serum Albumin

Various molecules can also be fused to the insulin polypeptides of theinvention to modulate the half-life of insulin polypeptides in serum. Insome embodiments, molecules are linked or fused to insulin polypeptidesof the invention to enhance affinity for endogenous serum albumin in ananimal.

For example, in some cases, a recombinant fusion of a insulinpolypeptide and an albumin binding sequence is made. Exemplary albuminbinding sequences include, but are not limited to, the albumin bindingdomain from streptococcal protein. G (see. e.g., Makrides et al., J.Pharmacol. Exp. Ther. 277:534-542 (1996) and Sjolander et al., J,Immunol. Methods 201:115-123 (1997)), or albumin-binding peptides suchas those described in, e.g., Dennis, et al., J. Biol. Chem.277:35035-35043 (2002).

In other embodiments, the insulin polypeptides of the present inventionare acylated with fatty acids. In some cases, the fatty acids promotebinding to serum albumin. See, e.g., Kurtzhals, et al., Biochem. J.312:725-731 (1995).

In other embodiments, the insulin polypeptides of the invention arefused directly with serum albumin (including but not limited to, humanserum albumin). Those of skill in the art will recognize that a widevariety of other molecules can also be linked to insulin in the presentinvention to modulate binding to serum albumin or other serumcomponents.

Glycosylation of Insulin Polypeptides

The invention includes insulin polypeptides incorporating one or morenon-naturally encoded amino acids bearing saccharide residues. Thesaccharide residues may be either natural (including but not limited to,N-acetylglucosamine) or non-natural including but not limited to,3-fluorogalactose). The saccharides may be linked to the non-naturallyencoded amino acids either by an N- or O-linked glycosidic linkage(including but not limited to, N-acetylgalactose-L-serine) or anon-natural linkage (including but not limited to, an oxime or thecorresponding C- or S-linked glycoside).

The saccharide (including but not limited to, glycosyl) moieties can beadded to insulin polypeptides either in vivo or in vitro. In someembodiments of the invention, an insulin polypeptide comprising acarbonyl-containing non-naturally encoded amino acid is modified with asaccharide derivatized with an aminooxy group to generate thecorresponding glycosylated polypeptide linked via an oxime linkage. Onceattached to the non-naturally encoded amino acid, the saccharide may befurther elaborated by treatment with glycosyltransferases and otherenzymes to generate an oligosaccharide bound to the insulin polypeptide.See, e.g., H. Liu, et al. J. Am. Chem. Soc. 125: 1702-1703 (2003).

In some embodiments of the invention, an insulin polypeptide comprisinga carbonyl-containing non-naturally encoded amino acid is modifieddirectly with a glycan with defined structure prepared as an aminooxyderivative. One of ordinary skill in the art will recognize that otherfunctionalities, including azide, alkyne, hydrazide, hydrazine, andsemicarbazide, can be used to link the saccharide to the non-naturallyencoded amino acid.

In some embodiments of the invention, an insulin polypeptide comprisingan azide or alkynyl-containing non-naturally encoded amino acid can thenbe modified by, including but not limited to, a Huisgen [3+2]cycloaddition reaction with, including but not limited to, alkynyl orazide derivatives, respectively. This method allows for proteins to bemodified with extremely high selectivity.

Insulin Dimers and Multimers

The present invention also provides for insulin and insulin analogcombinations such as homodimers, heterodimers, homomultimers, orheteromultimers (i.e., trimers, tetramers, etc.) where insulincontaining one or more non-naturally encoded amino acids is bound toanother insulin or insulin variant thereof or any other polypeptide thatis not insulin or insulin variant thereof, either directly to thepolypeptide backbone or via a linker. Due to its increased molecularweight compared to monomers, the insulin dimer or multimer conjugatesmay exhibit new or desirable properties, including but not limited todifferent pharmacological, pharmacokinetic, pharmacodynamic, modulatedtherapeutic half-life, or modulated plasma half-life relative to themonomeric insulin. For examples of monomeric insulin analogs see, forexample, Balschmidt, P., et al., U.S. Pat. No. 5,164,366, issued Nov.17, 1992; Brange, J., et al., U.S. Pat. No. 5,618,913, issued Apr. 8,1997; Chance, R. E., et al., U.S. Pat. No. 5,514,646, issued May 7,1996; and Ertl, J., et al., EPO publication number 885,961, Dec., 23,1998. Some embodiments of the present invention provide monomericinsulin analogs containing one or more non-naturally encoded amino acidresidues and in some embodiments these include monomeric insulin analogswherein position B28 is Asp, Lys, Ile, Leu, Val or Ala and the aminoacid residue at position B29 is Lys or Pro; monomeric insulin analogwith Lys(B28)Pro(B29)-human insulin; monomeric insulin analogAsp(B28)-human insulin; and monomeric insulin analogLys(B3)Ile(B28)-human insulin. In some embodiments, insulin dimers ofthe invention will modulate signal transduction of the insulin receptor.In other embodiments, the insulin dimers or multimers of the presentinvention will act as an insulin receptor antagonist, agonist, ormodulator.

In some embodiments, one or more of the insulin molecules present in aninsulin containing dimer or multimer comprises a non-naturally encodedamino acid linked to a water soluble polymer.

In some embodiments, the insulin polypeptides are linked directly,including but not limited to, via an Asn-Lys amide linkage or Cys-Cysdisulfide linkage. In some embodiments, the insulin polypeptides, and/orthe linked non-insulin molecule, will comprise different non-naturallyencoded amino acids to facilitate dimerization, including but notlimited to, an alkyne in one non-naturally encoded amino acid of a firstinsulin polypeptide and an azide in a second non-naturally encoded aminoacid of a second molecule will be conjugated via a Huisgen [3+2]cycloaddition. Alternatively, insulin, and/or the linked non-insulinmolecule comprising a ketone-containing non-naturally encoded amino acidcan be conjugated to a second polypeptide comprising ahydroxylamine-containing non-naturally encoded amino acid and thepolypeptides are reacted via formation of the corresponding oxime.

Alternatively, the two insulin polypeptides, and/or the linkednon-insulin molecule, are linked via a linker. Any hetero- orhomo-bifunctional linker can be used to link the two molecules, and/orthe linked non-insulin molecules, which can have the same or differentprimary sequence. In some cases, the linker used to tether the insulin,and/or the linked non-insulin molecules together can be a bifunctionalPEG reagent. The linker may have a wide range of molecular weight ormolecular length. Larger or smaller molecular weight linkers may be usedto provide a desired spatial relationship or conformation betweeninsulin and the linked entity or between insulin and its receptor, orbetween the linked entity and its binding partner, if any. Linkershaving longer or shorter molecular length may also be used to provide adesired space or flexibility between insulin and the linked entity, orbetween the linked entity and its binding partner, if any.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

In some embodiments, the invention provides multimers comprising one ormore insulin polypeptide, formed by reactions with water solubleactivated polymers that have the structure:

R—(CH2CH2O)n-O—(CH2)m-X

wherein n is from about 5 to 3,000, m is 2-10, X can be an azide, analkyne, a hydrazine, a hydrazide, an aminooxy group, a hydroxylamine, anacetyl, or carbonyl-containing moiety, and R is a capping group, afunctional group, or a leaving group that can be the same or differentas X. R can be, for example, a functional group selected from the groupconsisting of hydroxyl, protected hydroxyl, alkoxyl,N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester,N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal,aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, and ketone.

Measurement of Insulin Polypeptide Activity and Affinity of InsulinPolypeptide for the Insulin Receptor

Insulin polypeptide activity can be determined using standard or knownin vitro or in vivo assays. Insulin polypeptides may be analyzed forbiological activity by suitable methods known in the art. Such assaysinclude, but are not limited to, activation of interferon-responsivegenes, receptor binding assays, anti-viral activity assays, cytopathiceffect inhibition assays, (Familletti et. al., Meth. Enzymol.78:387-394), anti-proliferative assays, (Aebersold and Sample, Meth.Enzymol. 119:579-582), immunomodulatory assays (U.S. Pat. Nos.4,914,033; 4,753,795), and assays that monitor the induction of MHCmolecules (e.g., Hokland et al, Meth. Enzymol. 119:688-693), asdescribed in Meager, J. Immunol. Meth., 261:21-36 (2002).

Glucose uptake in 3T3-1 adipocytes may be assessed using the followingmethod. 3T3-L1 cells are obtained from the American Type CultureCollection (ATCC, Rockville, Md.). Cells are cultured in growth medium(GM) containing 10% iron-enriched fetal bovine serum in Dulbecco'smodified Eagle's medium. For standard adipocyte differentiation, 2 daysafter cells reached confluency (referred as day 0), cells are exposed todifferentiation medium (DM) containing 10% fetal bovine serum, 10 μg/mlof insulin, 1 μM dexamethasone, and 0.5 μM isobutylmethylxanthine, for48 hours. Cells then are maintained in post differentiation mediumcontaining 10% fetal bovine serum, and 10 μg/ml of insulin. In vitropotency may be measured with the glucose uptake assays which are knownto those of ordinary skill in the art. In vitro potency can be definedas the measure of glucose uptake of an insulin compound in a cell-basedassay and is a measure of the biological potency of the insulincompound. It can be expressed as the EC50 which is the effectiveconcentration of compound that results in 50% activity in a singledose-response experiment.

Glucose Transport Assay—Insulin Dependent—Hexose uptake, as assayed bythe accumulation of 0.1 mM 2-deoxy-D-[14C]glucose, is measured asfollows: 3T3-L1 adipocytes in 12-well plates are washed twice with KRPbuffer (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO4, 0.9 mM CaCl2, 0.9 mMMgSO4, pH 7.4) warmed to 37° C. and containing 0.2% BSA, incubated inLeibovitz's L-15 medium containing 0.2% BSA for 2 hours at 37° C. inroom air, washed twice again with KRP containing, 0.2% BSA buffer, andincubated in KRP 0.2% BSA buffer in the absence (Me2SO only) or presenceof wortmannin for 30 minutes at 37° C. in room air. Insulin is thenadded to a final concentration of 100 nM for 15 minutes, and the uptakeof 2-deoxy-D-[14C]glucose is measured for the last 4 minutes.Nonspecific uptake, measured in the presence of 10 μM cytochalasin B, issubtracted from all values. Protein concentrations are determined withthe Pierce bicinchoninic acid assay. Uptake is measured routinely intriplicate or quadruplicate for each experiment. The effect of acute andchronic pretreatment of 3T3-L1 adipocytes with FGF-21 in the presence ofinsulin may be investigated.

Glucose Transport Assay—Insulin Independent—3T3-L1 fibroblast are platedin 96-well plates and differentiated into fat cells (adipocytes) for 2weeks. After differentiation they are starved in serum-free medium andtreated with various insulin polypeptides of the present invention for24 hours. Upon treatment, cells are washed twice with KRBH buffer,containing 0.1% BSA. Glucose uptake is performed in the presence oflabeled glucose in KPBH buffer. This allows qualitative evaluation of avariety of insulin polypeptides and analogs produced by means of thepresent invention, and those which have been pegylated as pegylation hasbeen known to cause a decrease in efficiency of the native molecule, andcompare the efficacy of different insulins. Additionally, insulinpolypeptides of the present invention may be shown to induce glucoseuptake in an ex vivo tissue model.

In the ex vivo glucose transport model, the glucose transport assay isdescribed as follows: Krebs-Henseleit Buffer Stock Solutions—Stock 1:NaCl (1.16 M); KCl (0.046 M); KH2PO4 (0.0116 M); NaHCO3 (0.0253 M).Stock 2: CaCl2 (0.025 M); MgSO4 (2H2O) (0.0116 M). BSA: Use ICN CohnFraction V, fatty acid free BSA directly without dialysing. MediaPreparation: Add 50 ml of Krebs stock 1 to 395 ml of dH2O and gas with95% O2/5% CO2 for 1 hour. Add 50 ml of stock 2 and bring to 500 ml withdH2O. Add 500 mg of ICN fatty acid free BSA. Preincubation andIncubation Media: 32 mM Mannitol, 8 mM Glucos. Wash Media: 40 mMMannitol, 2 mM Pyruvate. Transport Media: 39 mM Mannitol, 1 mM 2-DG; 32mM Mannitol, 8 mM 3-O-MG. Insulin Solution: (Porcine Insulin [Lilly]100,000,000 μU/ml) at a final concentration of 2000 μU/ml or 13.3 nM.Radioactive Label Media Preparation: Specific activities used: 2DG=1.5mCi/ml; 3-O-MG=437 μCi/ml; or, Mannitol=8 μCi/m. Rats are anesthetizedwith 0.1 cc Nembutal per 100 g body weight. Muscle tissue is excised andrinsed in 0.9% saline then placed in pre-incubation media (2 ml) at 29°C. for 1 hour. The muscle tissue is transferred to incubation media (2ml; same as pre-incubation except including insulin or test compound)and incubated for 30 minutes (depends upon experimental conditions). Themuscle tissue is then transferred to wash media (2 ml) for 10 minutes at29° C., then transferred to label media (1.5 ml) for 10 min (3-O-MG) or20 min (2DG). The muscle tissue is trimmed, weighed and placed inpolypropylene tubes on dry ice. 1 ml of 1 N KOH is added to the tubeswhich are then placed in a 70° C. water bath for 10-15 minutes,vortexing the tubes every few minutes. The tubes are cooled on ice and 1ml of 1 N HCl is added, then mixed well. 200 μl of supernatant is thenput in duplicate scintillation vials and counted on a scintillationcounter compared to known radioactive standards.

For contraction, the muscles are first incubated for 1 hour inpreincubation/incubation media. After 1 hour, one muscle of each pair(one pair per rat) is pinned to the stimulation apparatus and the othermuscle is transferred to a new flask of incubation media. The contractedmuscle is stimulated by 200 msec trains of 70 Hz with each impulse in atrain being 0.1 msec. The trains are delivered at 1/see at 10-15V for2×10 minutes with a 1 minute rest in between. At the end of thestimulation period, the muscle is removed from the stimulation apparatusand placed in wash media for 10 minutes, followed by label media asoutlined above.

Regardless of which methods are used to create the present insulinanalogs, the analogs are subject to assays for biological activity.Tritiated thymidine assays may be conducted to ascertain the degree ofcell division. Other biological assays, however, may be used toascertain the desired activity. Insulin polypeptides may be analyzed fortheir antiviral activity and/or antiproliferative activity.Antiproliferative assays are known to those of ordinary skill of theart. Basu et al. in Bioconjugate Chem (2006) 17:618-630 describe ananti-proliferation assay using A549 cells and MTT to measureproliferation. Biological assays such as assaying for the ability toinhibit viral replication, also provides indication of insulin activity.Assays known to one of ordinary skill of the art may be also used toassess the biological activity and potential side effects of insulinpolypeptides of the invention.

Average quantities of insulin, insulin polypeptides, and/or insulinanalogues of the present invention may vary and in particular should bebased upon the recommendations and prescription of a qualifiedphysician. The exact amount of insulin, insulin polypeptides, and/orinsulin analogues of the present invention is a matter of preferencesubject to such factors as the exact type and/or severity of thecondition being treated, the condition of the patient being treated, aswell as the other ingredients in the composition. The invention alsoprovides for administration of a therapeutically effective amount ofanother active agent. The amount to be given may be readily determinedby one of ordinary skill in the art based upon therapy with insulin,available insulin therapies, and/or other insulin analogues.

Pharmaceutical compositions of the invention may be manufactured in aconventional manner.

EXAMPLES

The following examples are offered to illustrate, but do not limit theclaimed invention,

Example 1

This example describes one of the many potential sets of criteria forthe selection of sites of incorporation of non-naturally encoded aminoacids into insulin.

FIG. 1 shows the structure and sequence of insulin (NP_(—)000198—HumanProtein accession number), and the table below includes SEQ ID NO: 13with the full length sequence of human insulin, SEQ ID NO: 14 with thefull length human insulin sequence without the leader, SEQ ID NO: 1-12contain A chain and B chain sequences for insulin, lispro, aspart,glulisine, detemir, and glargine. Insulin polypeptides were generated bysubstituting a naturally encoded amino acid with a non-naturally encodedamino acid. Each polypeptide had one of the amino acids substituted withpara-acetylphenylalanine or with para-aminophenylalanine. Thepolypeptides generated lacked the leader sequence and were A/B chaininsulin polypeptides (SEQ ID NO. 1-14). Each of the polypeptidesgenerated had a non-naturally encoded amino acid substitution at one ofthe following positions 1, 5, 8, 9, 10, 12, 14, 15, 18, 19, and 21 ofSEQ ID NO: 1 and 1, 2, 3, 4, 17, 20, 21, 22, 25, 28, and 29 of SEQ IDNO: 2.

FIG. 2 shows the structure of human insulin that was labeled using thePyMOL software (DeLano Scientific; Palo Alto, Calif.) and some aminoacids corresponding to those substituted with para-acetylphenylalaninein insulin polypeptides of the invention. FIG. 6 shows the sequencehomology between insulin and known insulin analogues.

Another set of criteria for the selection of preferred sites ofincorporation of non-naturally encoded amino acids includes using andcomparing crystal structures from the Protein Data Bank, or other databanks, are used to model the structure of insulin and residues areidentified that 1) would not interfere with binding to the FGF receptoror heparin, and 2) would not be present in the interior of the protein.In some embodiments, one or more non-naturally encoded encoded aminoacids are incorporated at, but not limited to, one or more of thefollowing positions of insulin: 1, 5, 8, 9, 10, 12, 14, 15, 18, 19, and21 of SEQ ID NO: 1 and 1, 2, 3, 4, 17, 20, 21, 22, 25, 28, and 29 of SEQID NO: 2, In some embodiments, one or more non-naturally encoded encodedamino acids are incorporated at, but not limited to, one or more of thefollowing positions of insulin: 1, 5, 8, 9, 10, 12, 14, 15, 18, 19, and21 of SEQ ID NO: 1 (or the corresponding amino acids in SEQ ID NOs: 3,5, 7, 9, 11). In some embodiments, one or more non-naturally encodedencoded amino acids are incorporated at, but not limited to, one or moreof the following positions of insulin: 1, 2, 3, 4, 17, 20, 21, 22, 25,28, and 29 of SEQ ID NO: 2 (or the corresponding amino acids in SEQ IDNOs: 4, 6, 8, 10, 12). In some embodiments, one or more non-naturallyencoded encoded amino acids are incorporated at, but not limited to, oneor more of the following positions of insulin: 1, 5, 8, 9, 10, and 12 ofSEQ ID NO: 1 (or the corresponding amino acids in SEQ ID NOs: 3, 5, 7,9, 11). In some embodiments, one or more non-naturally encoded encodedamino acids are incorporated at, but not limited to, one or more of thefollowing positions of insulin: 1,2,3, 4, 17, and 20 of SEQ ID NO: 2 (orthe corresponding amino acids in SEQ ID NOs: 4, 6, 8, 10, 12). In someembodiments, one or more non-naturally encoded encoded amino acids areincorporated at, but not limited to, one or more of the followingpositions of insulin: 14, 15, 18, 19, and 21 of SEQ ID NO: 1 (or thecorresponding amino acids in SEQ ID NOs: 3, 5, 7, 9, 11). In someembodiments, one or more non-naturally encoded encoded amino acids areincorporated at, but not limited to, one or more of the followingpositions of insulin: 21, 22, 25, 28, and 29 of SEQ ID NO: 2 (or thecorresponding amino acids in SEQ ID NOs: 4, 6, 8, 10, 12).

The following criteria were used to evaluate each position of insulinand insulin analogs for the introduction of a non-naturally encodedamino acid: the residue (a) should not interfere with binding of theinsulin receptor based on structural analysis, b) should not be affectedby alanine or homolog scanning mutagenesis (c) should be surface exposedand exhibit minimal van der Waals or hydrogen bonding interactions withsurrounding residues, (d) should be either deleted or variable ininsulin variants, (e) would result in conservative changes uponsubstitution with a non-naturally encoded amino acid and (f) could befound in either highly flexible regions or structurally rigid regions.In addition, further calculations can be performed on the insulinmolecule, utilizing the Cx program (Pintar et al. (2002) Bioinformatics,18, pp 980) to evaluate the extent of protrusion for each protein atom.

In some embodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulin:before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 1 or the correspondingamino acids in SEQ ID NOs: 3, 5, 7, 9, 11). In some embodiments, one ormore non-naturally encoded amino acids are incorporated in one or moreof the following positions in insulin: before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 (i.e., at thecarboxyl terminus of the protein) (SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NOs: 4, 6, 8, 10, 12). In some embodiments, one ormore non-naturally encoded amino acids are incorporated in one or moreof the following positions in insulin: 8, 9, 10, 14 (SEQ ID NO: 1 or thecorresponding amino acids in SEQ ID NOs: 3, 5, 7, 9, 11). In someembodiments, one or more non-naturally encoded amino acids areincorporated in one or more of the following positions in insulin: 1,17, 25, 28 (SEQ ID NO: 2 or the corresponding amino acids in SEQ ID NOs:4, 6, 8, 10, 12).

Example 2

This example details cloning and expression of an insulin polypeptideincluding a non-naturally encoded amino acid in E. coli.

Methods for cloning insulin are known to those of ordinary skill in theart. Polypeptide and polynucleotide sequences for insulin and cloning ofinsulin into host cells are detailed in U.S. Pat. No. 5,962,267; U.S.Pat. Nos. 4,751,180, 7,105,314, 6,630,348, 6,777 and 7,091,032 Annibalipatent entitled “Expression of a human insulin precursor in P.pastoris”, as well as U.S. Patent Publication Numbers 20080268519,20080255045, 20080227205, 20080207877, 20080207877, 20080213828,20080261311, and 20080227195; all of which patents and publishedapplications are herein incorporated by reference.

cDNA encoding the lispro forms of insulin are shown as SEQ ID NOs: 34,35, 36, and 37. This mature polypeptide is shown as SEQ ID NOs: 3 and 4.

An introduced translation system that comprises an orthogonal tRNA(O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS) is used toexpress insulin or insulin analogs containing a non-naturally encodedamino acid. The O-RS preferentially aminoacylates the O-tRNA with anon-naturally encoded amino acid. In turn the translation system insertsthe non-naturally encoded amino acid into the insulin or insulin analog,in response to an encoded selector codon. Suitable O-RS and O-tRNAsequences are described in WO 2006/068802 entitled “Compositions ofAminoacyl-tRNA Synthetase and Uses Thereof” (E9; SEQ ID NO: 15) and WO2007/021297 entitled “Compositions of tRNA and Uses Thereof” (F13; SEQID NO: 16), which are incorporated by reference in their entiretyherein.

SEQ ID NO: 17 M. jannaschii mtRNA_(CUA) ^(Tyr) tRNA SEQ ID NO: 18HLAD03; an optimized amber supressor tRNA tRNA SEQ ID NO: 19 HL325A; anoptimized AGGA frameshift supressor tRNA tRNA SEQ ID NO: 20 AminoacyltRNA synthetase for the incorporation of p-azido-L-phenylalanine RSp-Az-PheRS(6) SEQ ID NO: 21 Aminoacyl tRNA synthetase for theincorporation of p-benzoyl-L-phenylalanine RS p-BpaRS(1) SEQ ID NO: 22Aminoacyl tRNA synthetase for the incorporation ofpropargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO: 23 Aminoacyl tRNAsynthetase for the incorporation of propargyl-phenylalanine RSPropargyl-PheRS SEQ ID NO: 24 Aminoacyl tRNA synthetase for theincorporation of propargyl-phenylalanine RS Propargyl-PheRS SEQ ID NO:25 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine RS p-Az-PheRS(1) SEQ ID NO: 26 Aminoacyl tRNAsynthetase for the incorporation of p-azido-phenylalanine RSp-Az-PheRS(3) SEQ ID NO: 27 Aminoacyl tRNA synthetase for theincorporation of p-azido-phenylalanine RS p-Az-PheRS(4) SEQ ID NO: 28Aminoacyl tRNA synthetase for the incorporation of p-azido-phenylalanineRS p-Az-PheRS(2) SEQ ID NO: 29 Aminoacyl tRNA synthetase for theincorporation of p-acetyl-phenylalanine (LW1) RS SEQ ID NO: 30 AminoacyltRNA synthetase for the incorporation of p-acetyl-phenylalanine (LW5) RSSEQ ID NO: 31 Aminoacyl tRNA synthetase for the incorporation ofp-acetyl-phenylalanine (LW6) RS SEQ ID NO: 32 Aminoacyl tRNA synthetasefor the incorporation of p-azido-phenylalanine (AzPheRS-5) RS SEQ ID NO:33 Aminoacyl tRNA synthetase for the incorporation ofp-azido-phenylalanine (AzPheRS-6) RS

The transformation of E. coli with plasmids containing the modifiedinsulin or insulin analog gene and the orthogonal aminoacyl tRNAsynthetase/tRNA pair (specific for the desired non-naturally encodedamino acid) allows the site-specific incorporation of non-naturallyencoded amino acid into the insulin polypeptide.

Wild type mature insulin is amplified by PCR from a cDNA synthesisreaction using standard protocols and cloned into pET30 (NcoI-BamHI).Prior to or alternatively following sequence confirmation, insulinincluding an N-terminal HHHHHHSGG sequence is subcloned into asuppression vector containing an amber suppressor tyrosyl tRNATyr/CUAfrom Methanococcus jannaschii (Mj tRNATyr/CUA) under constitutivecontrol of a synthetic promoter derived from the E. coli lipoproteinpromoter sequence (Miller, J. H., Gene, 1986), as well as well as theorthogonal tyrosyl-tRNA-synthetase (MjTyrRS) under control of the E.coli GlnRS promoter. Expression of insulin is under control of the T7promoter. Amber mutations are introduced using standard quick changemutation protocols (Stratagene; La Jolla, Calif.). Constructs aresequence verified.

Testing of long-acting insulin compounds may be done using the STZdiabetic rat model (PCO 08-400-209).

Suppression with Para-Acetyl-Phenylalanine (pAcF)

Plasmids (pVK6-LisPro Insulin) were transformed into the W3110 B2 strainof E. coli in which expression of the T7 polymerase was under control ofan arabinose-inducible promoter. Overnight bacterial cultures werediluted 1:100 into shake flasks containing 2×YT culture media and grownat 37° C. to an OD₆₀₀ of ˜0.8. Protein expression was induced by theaddition of arabinose (0.2% final), and para-acetyl-phenylalanine (pAcF)to a final concentration of 4 mM. Cultures were incubated at 37° C. for5 hours. Cells were pelleted and resuspended in B-PER lysis buffer(Pierce) 100 ul/OD/ml+10 ug/ml DNase and incubated at 37° C. for 30 min.Cellular material was removed by centrifugation and the supernatantremoved. The pellet was re-suspended in an equal amount of SDS-PAGEprotein loading buffer. All samples were loaded on a 4-12% PAGE gel withMES and DTT. Methods for purification of insulin are known to those ofordinary skill in the art and are confirmed by SDS-PAGE, Western Blotanalyses, or electrospray-ionization ion trap mass spectrometry and thelike.

The plasmid and sequence used are shown in FIG. 5, and expression ofN-terminal His tagged insulin and suppression at 7 amber sites is shownas FIG. 6. The proinsulin polypeptide is marked with an arrow. FIG. 6shows the B-PER pellet samples—to the left of Lane 1: Marker; Lane 1:VK6-proinsulin-pAF-Q15; Lane 2: VK6-proinsulin-pAF-Y14; Lane 3:VK6-proinsulin-pAF-R22; Lane 4: VK6-proinsulin-pAF-F1; Lane 5:VK6-proinsulin-pAF-G1; Lane 6: VK6-proinsulin-pAF-S9, 0.2% arabinose;Lane 7: VK6-proinsulin-pAF-K28. The position numbers indicated for theamino acid substitutions in Lanes 1, 2, 5, and 6 are based on LisProInsulin chain A, SEQ ID NO: 3, and the position numbers indicated forthe amino acid substitutions in Lanes 3, 4, and 7 are based on LisProInsulin chain B, SEQ ID NO: 4.

His-tagged mutant insulin proteins can be purified using methods knownto those of ordinary skill in the art. The ProBond Nickel-ChelatingResin (Invitrogen, Carlsbad, Calif.) may be used via the standardHis-tagged protein purification procedures provided by the manufacturer.Functional measurements of the proteins may be done through methodsknown in the art, methods provided within this application andincorporated references, and alternatively an ELISA on live cells can bedeveloped to assess insulin polypeptides of the invention.

Example 3

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of an insulinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 5,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:3, 5, 7, 9, 11) and each of the residues before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 (i.e., at thecarboxyl terminus of the protein of SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NOs: 4, 6, 8, 10, 12) is separately substitutedwith a non-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-acetyl-phenylalanine into insulin are SEQ ID NO: 1 and 2 (A and Bchains of insulin), and SEQ ID NO: 16 or 17 (muttRNA, M. jannaschii),and 15, 29, 30 or 31 (TyrRS LW1, 5, or 6) described in Example 2 above.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 3 and N is approximately 5,000 MW. The purifiedinsulin containing p-acetylphenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-insulin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 4

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of an insulinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 20,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 1 or the corresponding amino acids in SEQ ID NOs:3, 5, 7, 9, 11) and each of the residues before position 1 (i.e. at theN-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 (i.e., at thecarboxyl terminus of the protein of SEQ ID NO: 2 or the correspondingamino acids in SEQ ID NOs: 4, 6, 8, 10, 12) is separately substitutedwith a non-naturally encoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-aminophenylalanine into insulin are SEQ ID NO: 1 and 2 (A and B chainsof insulin), and SEQ ID NO: 16 or 17 (muttRNA, M. jannaschii), andsequences described above and incorporated for site-specificincorporation of p-aminophenylalanine.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 3 and N is approximately 20,000 MW. The purifiedinsulin containing p-aminophenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-insulin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 5

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of an insulinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 20,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 13) is separately substituted with a non-naturallyencoded amino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-aminophenylalanine into insulin are SEQ ID NO: 13, and SEQ ID NO: 16or 17 (muttRNA, M. jannaschii), and sequences described above andincorporated for site-specific incorporation of p-aminophenylalanine.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 3 and N is approximately 20,000 MW. The purifiedinsulin containing p-aminophenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-insulin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 6

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of an insulinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 20,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87(i.e., at the carboxyl terminus of the protein of SEQ ID NO: 14) isseparately substituted with a non-naturally encoded amino acid havingthe following structure:

The sequences utilized for site-specific incorporation ofp-aminophenylalanine into insulin are SEQ ID NO: 14, and SEQ ID NO: 16or 17 (muttRNA, M. jannaschii), and sequences described above andincorporated for site-specific incorporation of p-aminophenylalanine.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH12

where R is methyl, n is 3 and N is approximately 20,000 MW. The purifiedinsulin containing p-aminophenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-insulin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 7

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of an insulinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 30,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 13 or the corresponding positions in SEQ ID NOs:1-12 and 14) is separately substituted with a non-naturally encodedamino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-aminophenylalanine into insulin are SEQ ID NO: 13 (or SEQ ID NO: 1, 2,or 14), and SEQ ID NO: 16 or 17 (muttRNA, M. jannaschii), and sequencesdescribed above and incorporated for site-specific incorporation ofp-aminophenylalanine.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 3 and N is approximately 30,000 MW. The purifiedinsulin containing p-aminophenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-insulin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 8

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of an insulinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 40,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 13 or the corresponding positions in SEQ ID NOs:1-12 and 14) is separately substituted with a non-naturally encodedamino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-aminophenylalanine into insulin are SEQ ID NO: 13 (or SEQ ID NO: 1, 2,or 14), and SEQ ID NO: 16 or 17 (muttRNA, M. jannaschii), and sequencesdescribed above and incorporated for site-specific incorporation ofp-aminophenylalanine.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 3 and N is approximately 40,000 MW. The purifiedinsulin containing p-aminophenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J, Am. Chem. Soc. 1959, 81, pp 475). ThePEG-insulin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 9

This example details introduction of a carbonyl-containing amino acidand subsequent reaction with an aminooxy-containing PEG.

This Example demonstrates a method for the generation of an insulinpolypeptide that incorporates a ketone-containing non-naturally encodedamino acid that is subsequently reacted with an aminooxy-containing PEGof approximately 10,000 MW. Each of the residues before position 1 (i.e.at the N-terminus), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111 (i.e., at the carboxyl terminus of theprotein of SEQ ID NO: 13 or the corresponding positions in SEQ ID NOs:1-12 and 14) is separately substituted with a non-naturally encodedamino acid having the following structure:

The sequences utilized for site-specific incorporation ofp-aminophenylalanine into insulin are SEQ ID NO: 13 (or correspondingpositions in SEQ ID NO: 1, 2, or 14), and SEQ ID NO: 16 or 17 (muttRNA,M. jannaschii), and sequences described above and incorporated forsite-specific incorporation of p-aminophenylalanine.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with an aminooxy-containingPEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 3 and N is approximately 10,000 MW. The purifiedinsulin containing p-aminophenylalanine dissolved at 10 mg/mL in 25 mMMES (Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (SigmaChemical, St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (SigmaChemical, St. Louis, Mo.) pH 4.5, is reacted with a 10 to 100-foldexcess of aminooxy-containing PEG, and then stirred for 10-16 hours atroom temperature (Jencks, W. J. Am. Chem. Soc. 1959, 81, pp 475). ThePEG-insulin is then diluted into appropriate buffer for immediatepurification and analysis.

Example 10

Conjugation with a PEG consisting of a hydroxylamine group linked to thePEG via an amide linkage.

A PEG reagent having the following structure is coupled to aketone-containing non-naturally encoded amino acid using the proceduredescribed in Examples 3-9:

R-PEG(N)—O—(CH₂)₂—NH—C(O)(CH₂)_(n)—O—NH₂

where R=methyl, n=4 and N is approximately 5,000 MW-40,000 MW. Thereaction, purification, and analysis conditions are as described andknown in the art.

Example 11

This example details the introduction of two distinct non-naturallyencoded amino acids into insulin polypeptides and insulin analogpolypeptides.

This example demonstrates a method for the generation of an insulinpolypeptide that incorporates non-naturally encoded amino acidcomprising a ketone functionality at two positions among the followingresidues: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111(i.e., at the carboxyl terminus of the protein of SEQ ID NO: 13 or thecorresponding positions in SEQ ID NOs: 1-12 and 14). The insulinpolypeptide is prepared as described above, except that the selectorcodon is introduced at two distinct sites within the nucleic acid.

Example 12

This example details conjugation of insulin polypeptide or insulinanalog polypeptide to a hydrazide-containing PEG and subsequent in situreduction.

An insulin polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described above. Once modified, ahydrazide-containing PEG having the following structure is conjugated tothe insulin polypeptide:

R-PEG(N)—O—(CH2)2-NH—C(O)(CH2)n-X—NH—NH2

where R=methyl, n=2 and N=5,000; 10,000, 20,000; 30,000; or 40,000 MWand X is a carbonyl (C═O) group. The purified insulin containingp-acetylphenylalanine is dissolved at between 0.1-10 mg/mL in 25 mM MES(Sigma Chemical, St. Louis, Mo.) pH 6.0, 25 mM Hepes (Sigma Chemical,St. Louis, Mo.) pH 7.0, or in 10 mM Sodium Acetate (Sigma Chemical, St.Louis, Mo.) pH 4.5, is reacted with a 1 to 100-fold excess ofhydrazide-containing PEG, and the corresponding hydrazone is reduced insitu by addition of stock 1M NaCNBH3 (Sigma Chemical, St. Louis, Mo.),dissolved in H2O, to a final concentration of 10-50 mM. Reactions arecarried out in the dark at 4□C to RT for 18-24 hours. Reactions arestopped by addition of 1 M Tris (Sigma Chemical, St. Louis, Mo.) atabout pH 7.6 to a final Tris concentration of 50 mM or diluted intoappropriate buffer for immediate purification.

Example 13

This example details conjugation of insulin polypeptide or insulinanalog polypeptide to a hydrazide-containing PEG and subsequent in situreduction.

An insulin polypeptide incorporating a carbonyl-containing amino acid isprepared according to the procedure described above. Once modified, ahydrazide-containing PEG having the following structure is conjugated tothe insulin polypeptide:

R-PEG(N)—O—(CH2)2-NH—C(O)(CH2)n-X—NH—NH2

where R=methyl, n=2 and N=20,000 MW and X is a carbonyl (C═O) group. Thepurified insulin containing p-acetylphenylalanine is dissolved atbetween 0.1-10 mg/mL in 25 mM MES (Sigma Chemical, St. Louis, Mo.) pH6.0, 25 mM Hepes (Sigma Chemical, St. Louis, Mo.) pH 7.0, or in 10 mMSodium Acetate (Sigma Chemical, St. Louis, Mo.) pH 4.5, is reacted witha 1 to 100-fold excess of hydrazide-containing PEG, and thecorresponding hydrazone is reduced in situ by addition of stock 1MNaCNBH3 (Sigma Chemical, St. Louis, Mo.), dissolved in H2O, to a finalconcentration of 10-50 mM. Reactions are carried out in the dark at 4°C. to RT for 18-24 hours. Reactions are stopped by addition of 1 M Tris(Sigma Chemical, St. Louis, Mo.) at about pH 7.6 to a final Trisconcentration of 50 mM or diluted into appropriate buffer for immediatepurification.

Example 14

This example details introduction of an alkyne-containing amino acidinto an insulin polypeptide or insulin analog polypeptide andderivatization with mPEG-azide.

The following residues, before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111 (i.e., at the carboxyl terminus of the protein of SEQ IDNO: 13 or the corresponding positions in SEQ ID NOs: 1-12 and 14), areeach substituted with the following non-naturally encoded amino acid:

The sequences utilized for site-specific incorporation ofp-propargyl-tyrosine into insulin are SEQ ID NO: 13 (or correspondingpositions in SEQ ID NO: 1, 2, or 14), SEQ ID NO: 16 or 17 (muttRNA, M.jannaschii), and 22, 23 or 24 described above. The insulin polypeptidecontaining the propargyl tyrosine is expressed in E. coli and purifiedusing the conditions described above.

The purified insulin containing propargyl-tyrosine dissolved at between0.1-10 mg/mL in PB buffer (100 mM sodium phosphate, 0.15 M NaCl, pH=8)and a 10 to 1000-fold excess of an azide-containing PEG is added to thereaction mixture. A catalytic amount of CuSO4 and Cu wire are then addedto the reaction mixture. After the mixture is incubated (including butnot limited to, about 4 hours at room temperature or 37° C., orovernight at 4° C.), H2O is added and the mixture is filtered through adialysis membrane. The sample can be analyzed for the addition,including but not limited to, by similar procedures described in Example3. In this Example, the PEG will have the following structure

R-PEG(N)—O—(CH2)2-NH—C(O)(CH2)n-N3

where R is methyl, n is 4 and N=5,000; 10,000, 20,000; 30,000; or 40,000MW.

Example 15

This example details substitution of a large, hydrophobic amino acid inan insulin polypeptide with propargyl tyrosine.

A Phe, Trp or Tyr residue present within one the following regions ofinsulin: before position 1 (i.e. at the N-terminus), 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111(i.e., at the carboxyl terminus of the protein of SEQ ID NO: 13 or thecorresponding positions in SEQ ID NOs: 1-12 and 14), is substituted withthe following non-naturally encoded amino acid as described above:

Once modified, a PEG is attached to the insulin polypeptide variantcomprising the alkyne-containing amino acid. The PEG will have thefollowing structure:

Me-PEG(N)—O—(CH2)2-N3

and coupling procedures would follow those in examples above. This willgenerate an insulin polypeptide variant comprising a non-naturallyencoded amino acid that is approximately isosteric with one of thenaturally-occurring, large hydrophobic amino acids and which is modifiedwith a PEG derivative at a distinct site within the polypeptide.

Example 16

This example details generation of an insulin polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG linkers. Insulin polypeptide multimers may be formed betweenproinsulins or between mature A and B chain insulin polypeptides of theinvention.

The alkyne-containing insulin polypeptide variant produced in theexample above is reacted with a bifunctional PEG derivative of the form:

N3-(CH2)n-C(O)—NH—(CH2)2-O-PEG(N)—O—(CH2)2-NH—C(O)—(CH2)n-N3

where n is 4 and the PEG has an average MW of approximately 5,000;10,000; 20,000; 30,000; or 40,000 MW to generate the correspondinginsulin polypeptide homodimer where the two insulin molecules arephysically separated by PEG. In an analogous manner an insulinpolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in the examples above.

Example 17

This example details generation of an insulin polypeptide homodimer,heterodimer, homomultimer, or heteromultimer separated by one or morePEG linkers. Insulin polypeptide multimers may be formed between Achains and other A chains or B chains and other B chains.

The alkyne-containing insulin polypeptide variant produced in theexample above is reacted with a bifunctional PEG derivative of the form:

N3-(CH2)n-C(O)—NH—(CH2)2-O-PEG(N)—O—(CH2)2-NH—C(O)—(CH2)n-N3

where n is 4 and the PEG has an average MW of approximately 5,000;10,000; 20,000; 30,000; or 40,000 MW to generate the correspondinginsulin polypeptide homodimer where the two insulin molecules arephysically separated by PEG. In an analogous manner an insulinpolypeptide may be coupled to one or more other polypeptides to formheterodimers, homomultimers, or heteromultimers. Coupling, purification,and analyses will be performed as in the examples above.

Example 18

This example details coupling of a saccharide moiety to an insulinpolypeptide.

One residue of the following is substituted with the non-naturallyencoded amino acid below: before position 1 (i.e. at the N-terminus), 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111 (i.e., at the carboxyl terminus of the protein of SEQ IDNO: 13 or the corresponding positions in SEQ ID NOs: 1-12 and 14) asdescribed above.

Once modified, the insulin polypeptide variant comprising thecarbonyl-containing amino acid is reacted with a β-linked aminooxyanalogue of N-acetylglucosamine (GlcNAc). The insulin polypeptidevariant (10 mg/mL) and the aminooxy saccharide (21 mM) are mixed inaqueous 100 mM sodium acetate buffer (pH 5.5) and incubated at 37° C.for 7 to 26 hours. A second saccharide is coupled to the firstenzymatically by incubating the saccharide-conjugated insulinpolypeptide (5 mg/mL) with UDP-galactose (16 nM) andβ-1,4-galacytosyltransferase (0.4 units/mL) in 150 mM HEPES buffer (pH7,4) for 48 hours at ambient temperature (Schanbacher et al. J. Biol.Chem. 1970, 245, 5057-5061).

Example 19

This example details generation of a PEGylated insulin polypeptideantagonist.

A residue, including but not limited to, those involved in insulinreceptor binding is substituted with the following non-naturally encodedamino acid as described above. Once modified, the insulin polypeptidevariant comprising the carbonyl-containing amino acid will be reactedwith an aminooxy-containing PEG derivative of the form:

R-PEG(N)—O—(CH2)n-O—NH2

where R is methyl, n is 4 and N is 5,000; 10,000; 20,000; 30,000; or40,000 MW to generate an insulin polypeptide antagonist comprising anon-naturally encoded amino acid that is modified with a PEG derivativeat a single site within the polypeptide. Coupling, purification, andanalyses are performed as described above.

Example 20

Generation of an insulin polypeptide homodimer, heterodimer,homomultimer, or heteromultimer in which the insulin molecules arelinked directly

An insulin polypeptide variant comprising the alkyne-containing aminoacid can be directly coupled to another insulin polypeptide variantcomprising the azido-containing amino acid. In an analogous manner aninsulin polypeptide polypeptide may be coupled to one or more otherpolypeptides to form heterodimers, homomultimers, or heteromultimers.More description regarding multimers which may be formed is providedabove in Examples 16 and 17 and coupling, purification, and analyses areperformed as described above.

Example 21

PEG-OH+Br—(Ch₂)_(n)—C≡CR′→PEG-O—(CH₂)_(n)—C≡CR′

A B

The polyalkylene glycol (P—OH) is reacted with the alkyl halide (A) toform the ether (B). In these compounds, n is an integer from one to nineand R′ can be a straight- or branched-chain, saturated or unsaturatedC1, to C20 alkyl or heteroalkyl group. R′ can also be a C3 to C7saturated or unsaturated cyclic alkyl or cyclic heteroalkyl, asubstituted or unsubstituted aryl or heteroaryl group, or a substitutedor unsubstituted alkaryl (the alkyl is a C1 to C20 saturated orunsaturated alkyl) or heteroalkaryl group. Typically, PEG-OH ispolyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG)having a molecular weight of 800 to 40,000 Daltons (Da).

Example 22

mPEG-OH+Br—CH₂—C≡CH→mPEG-O—CH₂—C≡CH

mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g, 0.1mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL). Asolution of propargyl bromide, dissolved as an 80% weight solution inxylene (0.56 mL, 5 mmol, 50 equiv., Aldrich), and a catalytic amount ofKI were then added to the solution and the resulting mixture was heatedto reflux for 2 hours. Water (1 mL) was then added and the solvent wasremoved under vacuum. To the residue was added CH2Cl2 (25 mL) and theorganic layer was separated, dried over anhydrous Na2SO4, and the volumewas reduced to approximately 2 mL. This CH2Cl2 solution was added todiethyl ether (150 mL) drop-wise. The resulting precipitate wascollected, washed with several portions of cold diethyl ether, and driedto afford propargyl-O-PEG.

Example 23

mPEG-OH+Br−(CH₂)₃—C≡CH→mPEG-O—(CH₂)₃—C≡CH

The mPEG-OH with a molecular weight of 20,000 Da (mPEG-OH 20 kDa; 2.0 g,0.1 mmol, Sunbio) was treated with NaH (12 mg, 0.5 mmol) in THF (35 mL).Fifty equivalents of 5-bromo-1-pentyne (0.53 mL, 5 mmol, Aldrich) and acatalytic amount of KI were then added to the mixture. The resultingmixture was heated to reflux for 16 hours. Water (1 mL) was then addedand the solvent was removed under vacuum. To the residue was addedCH2Cl2 (25 mL) and the organic layer was separated, dried over anhydrousNa2SO4, and the volume was reduced to approximately 2 mL. This CH2Cl2solution was added to diethyl ether (150 mL) drop-wise. The resultingprecipitate was collected, washed with several portions of cold diethylether, and dried to afford the corresponding alkyne. 5-chloro-1-pentynemay be used in a similar reaction.

Example 24

m-HOCH₂C₆H₄OH+NaOH+Br−CH₂—C≡CH→m-HOCH₂C₆H₄O—CH₂—C≡CH  (1)

m-HOCH₂C₆H₄O—CH₂—C≡CH+MsCl+N(Et)₃ →m-MsOCH₂C₆H₄O—CH₂—C≡CH  (2)

m-MsOCH₂C₆H₄O—CH₂—C≡CH+LiBr→m-Br—CH₂C₆H₄O—CH₂—C≡CH  (3)

mPEG-OH+m-Br—CH₂C₆H₄O—CH₂—C≡CH→mPEG-O—CH₂—C₆H₄O—CH₂—C≡CH  (4)

To a solution of 3-hydroxybenzylalcohol (2.4 g, 20 mmol) in THF (50 mL)and water (2.5 mL) was first added powdered sodium hydroxide (1.5 g,37.5 mmol) and then a solution of propargyl bromide, dissolved as an 80%weight solution in xylene (3.36 mL, 30 mmol). The reaction mixture washeated at reflux for 6 hours. To the mixture was added 10% citric acid(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over MgSO4 andconcentrated to give the 3-propargyloxybenzyl alcohol.

Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of compound 3 (2.0 g, 11.0 mmol) inCH2Cl2 at 0° C. and the reaction was placed in the refrigerator for 16hours. A usual work-up afforded the mesylate as a pale yellow oil. Thisoil (2.4 g, 9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g, 23.0mmol) was added. The reaction mixture was heated to reflux for 1 hourand was then cooled to room temperature. To the mixture was added water(2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa2SO4, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (1.0 g, 0.05 mmol, Sunbio) was dissolved in THF (20 mL)and the solution was cooled in an ice bath. NaH (6 mg, 0.25 mmol) wasadded with vigorous stirring over a period of several minutes followedby addition of the bromide obtained from above (2.55 g, 11.4 mmol) and acatalytic amount of KI. The cooling bath was removed and the resultingmixture was heated to reflux for 12 hours. Water (1.0 mL) was added tothe mixture and the solvent was removed under vacuum. To the residue wasadded CH2Cl2 (25 mL) and the organic layer was separated, dried overanhydrous Na2SO4, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in a whiteprecipitate, which was collected to yield the PEG derivative.

Example 25

mPEG-NH₂+X—C(O)—(CH₂)_(n)—C≡CR′→mPEG-NH—C(O)—(CH₂)_(n)—C≡CR′

The terminal alkyne-containing poly(ethylene glycol) polymers can alsobe obtained by coupling a poly(ethylene glycol) polymer containing aterminal functional group to a reactive molecule containing the alkynefunctionality as shown above. n is between 1 and 10. R′ can be H or asmall alkyl group from C1 to C4.

Example 26

HO₂C—(CH₂)₂—C≡CH+NHS+DCC→NHSO—C(O)—(CH₂)₂—C≡CH  (1)

mPEG-NH₂+NHSO—C(O)—(CH₂)₂C≡CH→mPEG-NH—C(O)—(CH₂)₂—C≡CH  (2)

4-pentynoic acid (2.943 g, 3.0 mmol) was dissolved in CH2Cl2 (25 mL).N-hydroxysuccinimide (3.80 g, 3.3 mmol) and DCC (4.66 g, 3.0 mmol) wereadded and the solution was stirred overnight at room temperature. Theresulting crude NHS ester 7 was used in the following reaction withoutfurther purification.

mPEG-NH2 with a molecular weight of 5,000 Da (mPEG-NH2, 1 g, Sunbio) wasdissolved in THF (50 mL) and the mixture was cooled to 4° C. NHS ester 7(400 mg, 0.4 mmol) was added portion-wise with vigorous stirring. Themixture was allowed to stir for 3 hours while warming to roomtemperature. Water (2 mL) was then added and the solvent was removedunder vacuum. To the residue was added CH2Cl2 (50 mL) and the organiclayer was separated, dried over anhydrous Na2SO4, and the volume wasreduced to approximately 2 mL. This CH2Cl2 solution was added to ether(150 mL) drop-wise. The resulting precipitate was collected and dried invacuo.

Example 27

This Example represents the preparation of the methane sulfonyl ester ofpolyethylene glycol), which can also be referred to as themethanesulfonate or mesylate of polyethylene glycol). The correspondingtosylate and the halides can be prepared by similar procedures.

mPEG-OH+CH₃SO₂Cl+N(Et)₃ →mPEG-O—SO₂CH₃ →mPEG-N₃

The mPEG-OH (MW=3,400, 25 g, 10 mmol) in 150 mL of toluene wasazeotropically distilled for 2 hours under nitrogen and the solution wascooled to room temperature. 40 mL of dry CH2Cl2 and 2.1 mL of drytriethylamine (15 mmol) were added to the solution. The solution wascooled in an ice bath and 1.2 mL of distilled methanesulfonyl chloride(15 mmol) was added dropwise. The solution was stirred at roomtemperature under nitrogen overnight, and the reaction was quenched byadding 2 mL of absolute ethanol. The mixture was evaporated under vacuumto remove solvents, primarily those other than toluene, filtered,concentrated again under vacuum, and then precipitated into 100 mL ofdiethyl ether. The filtrate was washed with several portions of colddiethyl ether and dried in vacuo to afford the mesylate.

The mesylate (20 g, 8 mmol) was dissolved in 75 ml of THF and thesolution was cooled to 4° C. To the cooled solution was added sodiumazide (1.56 g, 24 mmol). The reaction was heated to reflux undernitrogen for 2 hours. The solvents were then evaporated and the residuediluted with CH2Cl2 (50 mL). The organic fraction was washed with NaClsolution and dried over anhydrous MgSO4. The volume was reduced to 20 mland the product was precipitated by addition to 150 ml of cold dryether.

Example 28

N₃—C₆H₄—CO₂H→N₃—C₆H₄CH₂OH  (1)

N₃—C₆H₄CH₂OH→Br—CH₂—C₆H₄—N₃  (2)

mPEG-OH+Br—CH₂—C₆H₄—N₃ →mPEG-O—CH₂—C₆H₄—N₃  (3)

4-azidobenzyl alcohol can be produced using the method described in U.S.Pat. No. 5,998,595, which is incorporated by reference herein.Methanesulfonyl chloride (2.5 g, 15.7 mmol) and triethylamine (2.8 mL,20 mmol) were added to a solution of 4-azidobenzyl alcohol (1.75 g, 11.0mmol) in CH2Cl2 at 0° C. and the reaction was placed in the refrigeratorfor 16 hours. A usual work-up afforded the mesylate as a pale yellowoil. This oil (9.2 mmol) was dissolved in THF (20 mL) and LiBr (2.0 g,23.0 mmol) was added. The reaction mixture was heated to reflux for 1hour and was then cooled to room temperature. To the mixture was addedwater (2.5 mL) and the solvent was removed under vacuum. The residue wasextracted with ethyl acetate (3×15 mL) and the combined organic layerswere washed with saturated NaCl solution (10 mL), dried over anhydrousNa2SO4, and concentrated to give the desired bromide.

mPEG-OH 20 kDa (2.0 g, 0.1 mmol, Sunbio) was treated with NaH (12 mg,0.5 mmol) in THF (35 mL) and the bromide (3.32 g, 15 mmol) was added tothe mixture along with a catalytic amount of KI. The resulting mixturewas heated to reflux for 12 hours. Water (1.0 mL) was added to themixture and the solvent was removed under vacuum. To the residue wasadded CH2Cl2 (25 mL) and the organic layer was separated, dried overanhydrous Na2SO4, and the volume was reduced to approximately 2 mL.Dropwise addition to an ether solution (150 mL) resulted in aprecipitate, which was collected to yield mPEG-O—CH2-C6H4-N3.

Example 29

NH₂-PEG-O—CH₂CH₂CO₂H+N₃—CH₂CH₂CO₂—NHS→N₃—CH₂CH₂—C(O)NH-PEG-O—CH₂CH₂CO₂H

NH2-PEG-O—CH2CH2CO2H (MW 3,400 Da, 2.0 g) was dissolved in a saturatedaqueous solution of NaHCO3 (10 mL) and the solution was cooled to 0° C.3-azido-1-N-hydroxysuccinimido propionate (5 equiv.) was added withvigorous stirring. After 3 hours, 20 mL of H2O was added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H2SO4 and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH2Cl2(100 mL×3), dried over Na2SO4 and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the omega-carboxy-azide PEG derivative.

Example 30

mPEG-OMs+HC≡CLi→mPEG-OMs+mPEG-O—CH₂—CH₂—C≡C—H

To a solution of lithium acetylide (4 equiv.), prepared as known in theart and cooled to −78° C. in THF, is added dropwise a solution ofmPEG-OMs dissolved in THF with vigorous stirring. After 3 hours, thereaction is permitted to warm to room temperature and quenched with theaddition of 1 mL of butanol. 20 mL of H2O is then added and the mixturewas stirred for an additional 45 minutes at room temperature. The pH wasadjusted to 3 with 0.5 N H2SO4 and NaCl was added to a concentration ofapproximately 15 wt %. The reaction mixture was extracted with CH₂Cl₂(100 mL×3), dried over Na2SO4 and concentrated. After precipitation withcold diethyl ether, the product was collected by filtration and driedunder vacuum to yield the 1-(but-3-ynyloxy)-methoxypolyethylene glycol(mPEG).

Example 31

Azide- and acetylene-containing amino acids can be incorporatedsite-selectively into proteins using the methods described in L. Wang,et al., (2001), Science 292:498-500, J. W. Chin et al., Science301:964-7 (2003)), J. W. Chin et al., (2002), Journal of the AmericanChemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz, (2002),Chem Bio Chem 3(11):1135-1137; J. W. Chin, et al., (2002), PNAS UnitedStates of America 99:11020-11024: and, L. Wang, & P. G. Schultz, (2002),Chem. Comm., 1:1-11. Once the amino acids were incorporated, thecycloaddition reaction is carried out with 0.01 mM protein in phosphatebuffer (PB), pH 8, in the presence of 2 mM PEG derivative, 1 mM CuSO4,and ˜1 mg Cu-wire for 4 hours at 37° C.

Example 32

This example describes the synthesis of p-Acetyl-D,L-phenylalanine (pAF)and m-PEG-hydroxylamine derivatives.

The racemic pAF is synthesized using the previously described procedurein Zhang, Z., Smith, B. A. C., Wang, L., Brock, A., Cho, C. & Schultz,P. G., Biochemistry, (2003) 42, 6735-6746.

To synthesize the m-PEG-hydroxylamine derivative, the followingprocedures are completed. To a solution of (N-t-Boc-aminooxy)acetic acid(0.382 g, 2.0 mmol) and 1,3-Diisopropylcarbodiimide (0.16 mL, 1.0 mmol)in dichloromethane (DCM, 70 mL), which is stirred at room temperature(RT) for 1 hour, methoxy-polyethylene glycol amine (m-PEG-NH2, 7.5 g,0.25 mmol, Mt. 30 K, from BioVectra) and Diisopropylethylamine (0.1 mL,0.5 mmol) is added. The reaction is stirred at RT for 48 hours, and thenis concentrated to about 100 mL. The mixture is added dropwise to coldether (800 mL). The t-Boc-protected product precipitated out and iscollected by filtering, washed by ether 3×100 mL. It is further purifiedby re-dissolving in DCM (100 mL) and precipitating in ether (800 mL)twice. The product is dried in vacuum yielding 7.2 g (96%), confirmed byNMR and Nihydrin test.

The deBoc of the protected product (7.0 g) obtained above is carried outin 50% TFA/DCM (40 mL) at 0° C. for 1 hour and then at RT for 1.5 hour.After removing most of TFA in vacuum, the TFA salt of the hydroxylaminederivative is converted to the HCl salt by adding 4N HCl in dioxane (1mL) to the residue. The precipitate is dissolved in DCM (50 mL) andre-precipitated in ether (800 mL). The final product (6.8 g, 97%) iscollected by filtering, washed with ether 3×100 mL, dried in vacuum,stored under nitrogen. Other PEG (5K, 20K) hydroxylamine derivatives aresynthesized using the same procedure.

Example 33 In Vivo Studies of PEGylated Insulin

[779] PEG-Insulin, unmodified insulin and buffer solution areadministered to mice or rats. The results will show superior activityand prolonged half life of the PEGylated insulin of the presentinvention compared to unmodified insulin. Similarly, modified insulin,unmodified insulin, and buffer solution are administered to mice orrats.

Pharmacokinetic Analysis

An insulin polypeptide of the invention is administered by intravenousor subcutaneous routes to mice. The animals are bled prior to and attime points after dosing. Plasma is collected from each sample andanalyzed by radioimmunoassay. Elimination half-life can be calculatedand compared between insulin polypeptides comprising a non-naturallyencoded amino acid and wild-type insulin or various insulin analogpolypeptides of the invention. Similarly, insulin polypeptides of theinvention may be administered to cynomolgus monkeys. The animals arebled prior to and at time points after dosing. Plasma is collected fromeach sample and analyzed by radioimmunoassay.

Polypeptides of the invention may be administered to ZDF male rats(diabetic, fat rats; 8 weeks of age at beginning of study, CharlesRiver-GMI). Rats are fed Purina 5008 feed ad libitum. The following testgroups are set up: Saline; Insulin 4 U/day; insulin polypeptides of theinvention, 8 U/day Acute (Acute dosing group is dosed once and bled atT=0, 2, 4, 8, and 24 hours post dose); insulin polypeptides of theinvention, 6 U/day; insulin polypeptides of the invention, 4 U/day;insulin polypeptides of the invention, 3 U/day; insulin polypeptides ofthe invention, 2 U/day; Lean Saline; Lean Insulin 4 U/day, and groupsreceiving 6 U/day, 4 U/day, and 2 U/day of insulin polypeptides innon-diabetic rats. Lean groups represent non-diabetic, lean, ZDF rats.

Compounds are injected s.c. once per day, and then secondary groups withsame administration amounts are maintained with injections every otherday. Control rats are injected with vehicle (PBS; 0.1 ml). Following 7days of dosing, the animals are subjected to an oral glucose tolerancetest. Blood for glucose and triglycerides are collected by tail clipbleeding without anesthetic. Insulin polypeptides may reduce plasmaglucose levels in a dose-dependent manner. Also lean ZDF rats may betested for hypoglycemia after exposure to insulin polypeptides of theinvention when compared to rats dosed with wild type insulin.

ob/ob Obesity Model

The ob/ob mouse model is an animal model for hyperglycemia, insulinresistance, and obesity. Plasma glucose levels after treatment withinsulin polypeptide compared to vehicle and insulin control groups maybe measured in ob/ob mice. In this obesity model, the test groups ofmale ob/ob mice (7 weeks old) are injected with vehicle alone (PBS),insulin (4 U/day), or insulin polypeptides as described above,subcutaneously (0.1 ml, once a day and in other test groups once everyother day and in other test groups once per week) for fourteen days.Blood is collected by tail clip bleeding on days 1, 3, and 7, 9, 11, 14,one hour after the first compound injection, and plasma glucose levelsare measured using a standard protocol. Insulin polypeptides of theinvention stimulate glucose uptake if they reduce plasma glucose levelswhen compared to the vehicle control group. Triglyceride levels may becompared after treatment with insulin polypeptides of the inventioncompared to other molecules. The polypeptide may be administered themice via multiple doses, continuous infusion, or a single dose, etc.

Example 34

Insulin expression system using Novagen (inducible T7 promoter;described in detail in the pET System Manual, version 9, herebyincorporated by reference), expression vector pET30a and expressionstrain BL21(DE3).

2 mL of LB/Kanamycin (10 μg/ml) culture are inoculated with a sweep fromBL21 (DE3) plate transformed with the desired analog. This decreaseseffects caused by colony to colony variability in expression levels.This culture is grown overnight at 37° C. with vigorous shaking and thefollowing day, 10 ml LB/Kanamycin culture is inoculated with 1 ml fromthe overnight culture (OD600˜0.4-0.5). The remaining mL of the overnightculture may be frozen as glycerol stock.

10 mL of the grown culture is put at 37° C. and 250 rpm for 30-45 minuntil OD600 reaches 0.8-0.9. This is then induced with 1 mM IPTG (with 1mL that may be set aside as non-induced culture control) and harvestedusually 3-4 hours post-induction and analyzed on SDS-PAGE.

It is also possible to do a time-course of expression (e.g. points 1, 2,4, 6 hours post-induction and O/N) to determine the rate ofaccumulation, protein stability, etc.

Gel Analysis: at desired time point post-induction 1 mL is harvestedfrom the culture, the cells alre spun down, resuspended in 100 μl of2×SDS-PAGE, sonicated to reduce viscosity and 10 μl are run on SDS-PAGE.If desired, this can be compared to non-induced control or controlsand/or known positive control or standard and expression level may beestimated (e.g. good expression could be @>100 μg/ml). Western blotanalysis may also be used. It is also possible to set aside 4 ml of thecultures, prepare inclusion bodies (if expressing insoluble analogs) andobtain mass spec analysis on these to confirm the identity of theover-expressed protein.

For larger scale protein expression, >250 mL of LB/Kanamycin (10 μg/ml)are inoculated with 250 μL of frozen glycerol stock and grown overnight.The following day, 10×1 L LB/Kanamycin cultures are inoculated with 25mL from the overnight culture (OD600˜0.1).

1 L cultures are grown at 37° C. and 250 rpm ˜2 h until OD600 reaches0.8-0.9. This is then induced with 1 mM IPTG and harvested 4 hpost-induction or the following morning (harvest may use centrifugationfor 15 min at 4,000 rpm). The pellets are rinsed with 50 mM Tris-HCl, pH8.0 (50 ml per pellet+50 ml to rinse the bottle) if it is desired toreduce endotoxin and facilitate purification. Pellets are pooledtogether and spun again.

Example 35 Pichia Expression Study—DNA Prep, Electroporation, ExpressionProtocols

This example provides a protocol for the preparation of insulinpolypeptides of the present invention in Pichia. SEQ ID NOs: 34, 35, 36,and 37 were used, and FIG. 8 shows a plasmid used for cloning intoPichia and this or other modified plasmids may be used to obtain proteinexpression of insulin polypeptides in Pichia, modifications made to theplasmid using methods known in the art.

On day 1 of the protocol, there is an overnight digestion, typicallyusing 2U enzyme per μg DNA to be digested and 10 mL YPhyD culture isinoculated overnight in a 50 mL flask, shaking at 260 rpm at 30° C. fromthe glycerol stock.

DNA Preparation

DNA is precipitated by the addition first of 1/10^(th) volume sterile 3MNaOAc and then of 0.7 volumes sterile IPA and then the sample isvigorously mixed and the precipitation is continued overnight at −20° C.or at −70° C. until frozen. The DNA is then pelleted by centrifugation(benchtop centrifuge 14,000 rpm/10 minutes), supernatant removed, andthe pellet is washed using 500 μL of sterile 70% ETOH. Spin (bench-topcentrifuge 14,000 rpm/10 minutes) and decant supernatant and air drypellet for 15-20 minutes. Resuspend DNA pellet with sterile water to 1μg/μl and transform Pichia with 10 μg DNA.

Electroporation

Using overnight culture with OD₆₀₀, dilute in YPhyD to OD₆₀₀=0.2. Shakeculture at 260 rpm at 30° C. until OD₆₀₀ reaches 0.8-1.0. Collect cellsby centrifugation (4000 rpm/5 minutes). Decant medium, wash cells in 20mL ice cold sterile water, decant again and repeat. After water wash,wash pellet in 20 mL of ice-cold sterile 1 M sorbitol, decant, andresuspend washed cell pellet in 600 μL of 1 M cold sorbitol, then thismay be stored on ice.

From the washed cells, mix 50 μL with 10 μg linearized DNA in sterile1.5 mL eppendorf tube, mix gently and incubate on ice for 25 minutes.Transfer cell/DNA mixture to prechilled 0.2 cm cuvette using longpipette tips. Electroporate cells using BioRad GenePulsar II unit withthe following settings: 2000 V, 200 Ohms, 25 μFd (use single pulse) andimmediately add 0.5 mL YPhyD medium to the cuvette and mix by pipetting.Transfer entire contents to sterile round bottom tube and shake gently(200 rpm) for 30 minutes at 30° C. Plate and spread cells evenly andincubate plates, inverted, for 3 days at 30° C.

After three day incubation, pick colonies with a loop and inoculate 10ml BYPhyD media in a 50 ml flask and incubate for 3 days at 30° C. Countthe colonies on the 20 μl plates and record the average number and thenharvest cells, first by preparing 2 sets of cryovials labeled withstrain name and clone number, insulin (i.e. protein expressed), anddate. Transfer culture to 15 ml conical tube, take OD₆₀₀ of eachculture, dilute culture 1:50 or 1:20 in YPhyD medium. Save an alquot ofculture for glycerol stock. Then pellet yeast at 4000 rpm for 5 min atRT, transfer the supernatant to a new, labeled 15 mL conical tube, andstore at −20 or −80° C. until needed for analytical data.

Protein Expression Analysis

Run samples on 4-12% NuPAGE TB gel (Novex). SDS-PAGE reagents used fromInvitrogen, analyze by Western blot or Stained-gel analysis

Media Formulations Buffered Yeast Phytone Dextrose (BYPhyD)

Yeast Extract  10 g/L Phytone Peptone  20 g/L 1 M potassium phosphatebuffer (pH 6) 100 ml/L 10X YNB 100 mL/L 20% Dextrose 100 mL/L

Yeast Phytone Dextrose (YPhyD)

Yeast Extract  10 g/L Phytone Peptone  20 g/L 20% Dextrose 100 ml/L10×YNB (13.4% Yeast Nitrogen Base with Ammonium Sulfate without AminoAcids)

Yeast Nitrogen Base 134 g/L

Example 36 Insulin A21G Production

In this example, 4.0 L culture were fermented to produce 13.4 g wet cellpaste and an inclusion body preparation was performed with and withoutTriton-X100. 2.07 g wet inclusion bodies were produced in this manner,and solubilization and refolding followed. The inclusion bodies wereresuspended with 200 mL H₂O per gram of wet inclusion bodies (IBs) to afinal concentration of 3 mM and cysteine is added to the resuspension.IB's are then solubilized by pH increase to 11.5 for 1 hour at RT.Refolding was then allowed to occur by dropping the pH of thesolubilized material to 10.6±0.1 and stored at 2-8° C. for ˜72 hours andthese results are shown in FIG. 10. The refold reaction was stopped byaddition of HCl to a final pH of 3.0, 0.45 μM filtered and stored at2-8° C. until further processing.

The refolded protein is purified (as shown in FIG. 11) by increasing thepH of quenched refold to 8.0 with Tris base and directly loading onto aQ HP column. Conductivity of load in the instance shown was >3.5 mS/cm.Run conditions were (A) 20 mM Tris, 8.0; (B) 20 mM Tris, 8.0; 200 mMNaCl and there was 0-100% B over 30CV. The correctly refolded proinsulinwas pooled and 79 mg proinsulin was recovered.

Ultrafiltration/diafiltration (UF/DF) took place and precipitation wasperformed with 25 mM zinc, precipitated protein was resuspended toconcentration of 2 mg/mL with 20 mM NaOAc, 4.0, 30% ACN, 5 mM EDTA and20K PEG was added to a final molar ratio of 10:1 PEG to protein andallowed to incubate for 48-72 hours at 28° C.

PEG reaction was diluted 1:10 in 0.5×PEG buffer A, 0.22 μM filtered andrun over an SP 650S column. The Run Conditions were (A) 10 mM NaOAc,4.0, 1 mM EDTA; (B) 10 mM NaOAc, 4.0, 1 mM EDTA, 0.4M NaCl; 0-50% B over20CV and PEG samples formulated in 10 mM NaCitrate, 6.5; 150 mM NaCl andthis is shown in FIG. 12.

These methods were used to produce a variety of insulin polypeptideswith non-natural amino acids and a range of 0.1-22 mg for the endprotein amounts of the purified and PEGylated variants. ACN was found tohelp solubilize PEG/protein mixture in PEG reaction and zincprecipitation at pI facilitated concentrating in the presence of CAN.

Example 37 Testing of Insulin Polypeptides in the STZ Diabetic Rat Model

Rats were treated with single injection of two different insulinpolypeptides of the present invention: A14pAF-PEG-A21N LysPro Insulinand A14pAF-PEG-A21G LysPro Insulin, each at four different levels; 568nmol/kg; 94 nmol/kg; 56.8 nmol/kg; and 9.4 nmol/kg (3, 0.5, 0.3, 0.05mg/kg insulin content).

The blood glucose was then measured at 1, 2, 4, 8, 12, 24, 48, 72, 96,120 h post dose and the average area under the curve, measurements ofblood glucose from 0-120 hours for each of these, including a controlinjecting only vehicle, are shown in FIG. 9.

Example 38 Human Clinical Trial of the Safety and/or Efficacy ofPEGylated Insulin Comprising a Non-Naturally Encoded Amino Acid

Objective: To observe the safety and pharmacokinetics of subcutaneouslyadministered PEGylated recombinant human insulin comprising anon-naturally encoded amino acid.

Patients Eighteen healthy volunteers ranging between 20-40 years of ageand weighing between 60-90 kg are enrolled in the study. The subjectswill have no clinically significant abnormal laboratory values forhematology or serum chemistry, and a negative urine toxicology screen,HIV screen, and hepatitis B surface antigen. They should not have anyevidence of the following: hypertension; a history of any primaryhematologic disease; history of significant hepatic, renal,cardiovascular, gastrointestinal, genitourinary, metabolic, neurologicdisease; a history of anemia or seizure disorder; a known sensitivity tobacterial or mammalian-derived products, PEG, or human serum albumin;habitual and heavy consumer to beverages containing caffeine;participation in any other clinical trial or had blood transfused ordonated within 30 days of study entry; had exposure to insulin withinthree months of study entry; had an illness within seven days of studyentry; and have significant abnormalities on the pre-study physicalexamination or the clinical laboratory evaluations within 14 days ofstudy entry. All subjects are evaluable for safety and all bloodcollections for pharmacokinetic analysis are collected as scheduled, Allstudies are performed with institutional ethics committee approval andpatient consent.

Study Design: This will be a Phase I, single-center, open-label,randomized, two-period crossover study in healthy male volunteers.Eighteen subjects are randomly assigned to one of two treatment sequencegroups (nine subjects/group). Insulin is administered over two separatedosing periods as a bolus s.c. injection in the upper thigh usingequivalent doses of the PEGylated insulin comprising a non-naturallyencoded amino acid and the commercially available product chosen. Thedose and frequency of administration of the commercially availableproduct is as instructed in the package label. Additional dosing, dosingfrequency, or other parameter as desired, using the commerciallyavailable products may be added to the study by including additionalgroups of subjects. Each dosing period is separated by a 14-day washoutperiod. Subjects are confined to the study center at least 12 hoursprior to and 72 hours following dosing for each of the two dosingperiods, but not between dosing periods. Additional groups of subjectsmay be added if there are to be additional dosing, frequency, or otherparameter, to be tested for the PEGylated insulin as well. Theexperimental formulation of insulin is the PEGylated insulin comprisinga non-naturally encoded amino acid.

Blood Sampling: Serial blood is drawn by direct vein puncture before andafter administration of insulin. Venous blood samples (5 mL) fordetermination of serum insulin concentrations are obtained at about 30,20, and 10 minutes prior to dosing (3 baseline samples) and atapproximately the following times after dosing: 30 minutes and at 1, 2,5, 8, 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample isdivided into two aliquots. All serum samples are stored at −20° C. Serumsamples are shipped on dry ice. Fasting clinical laboratory tests(hematology, serum chemistry, and urinalysis) are performed immediatelyprior to the initial dose on day 1, the morning of day 4, immediatelyprior to dosing on day 16, and the morning of day 19.

Bioanalytical Methods: An ELISA kit is used for the determination ofserum insulin concentrations.

Safety Determinations: Vital signs are recorded immediately prior toeach dosing (Days 1 and 16), and at 6, 24, 48, and 72 hours after eachdosing. Safety determinations are based on the incidence and type ofadverse events and the changes in clinical laboratory tests frombaseline. In addition, changes from pre-study in vital signmeasurements, including blood pressure, and physical examination resultsare evaluated.

Data Analysis Post-dose serum concentration values are corrected forpre-dose baseline insulin concentrations by subtracting from each of thepost-dose values the mean baseline insulin concentration determined fromaveraging the insulin levels from the three samples collected at 30, 20,and 10 minutes before dosing. Pre-dose serum insulin concentrations arenot included in the calculation of the mean value if they are below thequantification level of the assay. Pharmacokinetic parameters aredetermined from serum concentration data corrected for baseline insulinconcentrations. Pharmacokinetic parameters are calculated by modelindependent methods on a Digital Equipment Corporation VAX 8600 computersystem using the latest version of the BIOAVL software. The followingpharmacokinetics parameters are determined: peak serum concentration(Cmax); time to peak serum concentration (tmax); area under theconcentration-time curve (AUC) from time zero to the last blood samplingtime (AUC0-72) calculated with the use of the linear trapezoidal rule;and terminal elimination half-life (t½), computed from the eliminationrate constant. The elimination rate constant is estimated by linearregression of consecutive data points in the terminal linear region ofthe log-linear concentration-time plot. The mean, standard deviation(SD), and coefficient of variation (CV) of the pharmacokineticparameters are calculated for each treatment. The ratio of the parametermeans (preserved formulation/non-preserved formulation) is calculated.

Safety Results: The incidence of adverse events is equally distributedacross the treatment groups. There are no clinically significant changesfrom baseline or pre-study clinical laboratory tests or blood pressures,and no notable changes from pre-study in physical examination resultsand vital sign measurements. The safety profiles for the two treatmentgroups should appear similar.

Pharmacokinetic Results: Mean serum insulin concentration-time profiles(uncorrected for baseline insulin levels) in all 18 subjects afterreceiving PEGylated insulin comprising a non-naturally encoded aminoacid at each time point measured. All subjects should have pre-dosebaseline insulin concentrations within the normal physiologic range.Pharmacokinetic parameters are determined from serum data corrected forpre-dose mean baseline insulin concentrations and the Cmax and tmax aredetermined. The mean tmax for the any clinical comparator(s) chosen issignificantly shorter than the tmax for the PEGylated insulin comprisingthe non-naturally encoded amino acid. Terminal half-life values aresignificantly shorter for the preclinical comparator(s) tested comparedwith the terminal half-life for the PEGylated insulin comprising anon-naturally encoded amino acid.

Although the present study is conducted in healthy male subjects,similar absorption characteristics and safety profiles would beanticipated in other patient populations; such as male or femalepatients with diabetes, male or female patients with cancer or chronicrenal failure, pediatric renal failure patients, patients in autologouspredeposit programs, or patients scheduled for elective surgery.

In conclusion, subcutaneously administered single doses of PEGylatedinsulin comprising non-naturally encoded amino acid will be safe andwell tolerated by healthy male subjects. Based on a comparativeincidence of adverse events, clinical laboratory values, vital signs,and physical examination results, the safety profiles of thecommercially available forms of insulin and PEGylated insulin comprisingnon-naturally encoded amino acid will be equivalent. The PEGylatedinsulin comprising non-naturally encoded amino acid potentially provideslarge clinical utility to patients and health care providers.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to those of ordinary skill in the artand are to be included within the spirit and purview of this applicationand scope of the appended claims. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

TABLE 1 Sequences Cited SEQ ID NO: Sequence Name Sequence 1 Insulin Achain, GIVEQCCTSICSLYQLENYCN amino acid sequence 2 Insulin B chain,FVNQHLCGSHLVEALYLVCGERGFFYTPKT amino acid sequence 3 Insulin lispro AGIVEQCCTSICSLYQLENYCN chain, amino acid sequence (Humalog) 4 Insulinlispro B FVNQHLCGSHLVEALYLVCGERGFFYTKPT chain, amino acid sequence(Humalog) 5 Insulin aspart A GIVEQCCTSICSLYQLENYCN chain, amino acidsequence (Novolog) 6 Insulin aspart B FVNQHLCGSHLVEALYLVCGERGFFYTDKTchain, amino acid sequence (Novolog) 7 Insulin glulisine AGIVEQCCTSICSLYQLENYCN chain, amino acid sequence (Apidra) 8 Insulinglulisine B FVkQHLCGSHLVEALYLVCGERGFFYTDeT chain, amino acid sequence(Apidra) 9 Insulin detemir A GIVEQCCTSICSLYQLENYCG chain, amino acidsequence (Levemir) 10 Insulin detemir B FVNQHLCGSHLVEALYLVCGERGFFYTDKchain, amino acid sequence (Levemir) 11 Insulin glargine AGIVEQCCTSICSLYQLENYCG chain, amino acid sequence (Lantus) 12 Insulinglargine B FVNQHLCGSHLVEALYLVCGERGFFYTPKTRR chain, amino acid sequence(Lantus) 13 Full Length MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHL InsulinAmino VEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGG Acid SequencePGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN with leader 14 Full LengthFVNQHLCGSHLVEALYLVCGERGFFYTPK Insulin AminoTRREAEDLQVGQVELGGGPGAGSLQPLAL Acid Sequence EGSLQKRGIVEQCCTSICSLYQLENYCNwithout leader

DNA Sequences of LisPro Insulin for expression in Pichia LisPro & LengthLength in aa Configuaration in bp DNA seq INS1 KR- 213aagcgtgaggaggctgaggctgaggctgagcctaag 69 EEAEAEAEPK-ttcgtcaaccaacacttgtgcggttctcacttgg SEQ B1-30-RRLQKR-ttgaggccctttacttggtttgcggtgagcgtggtttcttctacaccaag ID NO: A1-21 cctact cgtcgtttacaaaagcgt 34 ggtatcgttgagcaatgctgcacctctatctgctccttgtaccaattggagaactactgcaac taatga INS2 KR- B1-30- 183 Aagcgtttcgtcaaccaacacttgtgcggttctcacttgg 59 RRLQKR-A1-21ttgaggccctttacttggtttgcggtgagcgtggtttctt SEQ ctacaccaagcctact ID NO:cgtcgtttacaaaagcgt 35 ggtatc gttgagcaatgctgcacctctatctgctccttgtaccaattggagaactactgcaac taatga INS3 KR- B1-30-KR-A1- 171aagcgt ttcgtcaaccaacacttgtgcggttctcactt 55 21 ggttgaggccctttacttggtttgcggtgagcgtggtttcttct SEQ acaccaagcctact aagcgt ggtatcgttgagcaatgctID NO: gcacctctatctgctccttg taccaattggagaactactgcaac 36 taatga INS4 KR-201 aagcgtgaggaggctgaggctgaggctgagcctaag 65 EEAEAEAEPK-ttcgtc aaccaacacttgtgcggttc tcacttggttgaggccctttac SEQ B1-30-KR-A1-21ttggtttgcggtgagcgtggtttcttctacaccaagcctact aagcgt ID NO:ggtatcgttgagcaatgctgcacctctatctgctccttgtaccaattgga 37gaactactgcaac taatga

1. An insulin polypeptide comprising one or more non-naturally encodedamino acids.
 2. An insulin polypeptide wherein the A chain comprises oneor more non-naturally encoded amino acids.
 3. An insulin polypeptidewherein the B chain comprises one or more non-naturally encoded aminoacids.
 4. An insulin analog comprising one or more non-naturally encodedamino acids.
 5. A method of treating a patient with a metabolic disorderwherein the patient is administered a therapeutically effective amountof an insulin polypeptide comprising one or more non-naturally encodedamino acids.